Bacillus compositions and methods of use with ruminants

ABSTRACT

Bacillus  strains, compositions and methods are disclosed for reducing growth of microorganisms in a feed.  Bacillus  strains, compositions and methods are disclosed for providing beneficial effects to animals, including but not limited to increasing performance of the animal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 62/341,332 filed May 25, 2016, the entirety of which is incorporated by reference herein.

FIELD OF THE DISCLOSURE

The disclosure relates to compositions and methods of Bacillus strains. In one embodiment, the disclosure relates to Bacillus strains, compositions and methods for controlling the growth of microorganisms, for example in a feed or fodder. In one embodiment, the disclosure relates to Bacillus strains, compositions and methods for improving performance of an animal; and more particularly relates to a Bacillus strain based direct fed microbial for improving performance of a ruminant

BACKGROUND

Organisms of the genus Clostridium are gram-positive, anaerobic, endospore-forming bacteria. Clostridium are normal inhabitants of the soil and intestinal tract of animals including dairy cows and calves. Many species are ubiquitous on dairy farms commonly found in haylage, corn silage, straw, manure, colostrum and cattle bedding material. Clostridium growth is limited in fermented forages by reducing the pH to less than 5.0 but the organisms may survive for an extended period even in well fermented forages.

There are over 100 species of Clostridium recognized, some of which are known to cause enteric disease while others are nonpathogenic with a broad range of enzymatic function and industrial uses. The species recognized to cause enteric disease in animals include C. perfringens, C. septicum, C. sordelli and C. botulinum. Examples of diseases caused by these organisms include necrotic enteritis, hemorrhagic bowel syndrome (HBS), malignant edema, abomasal disease and botulism.

C. perfringens-mediated diseases are a significant cause of economic loss to livestock industries. In dairy production, HBS is among the leading causes of digestive deaths and was reported to be responsible for at least 2% of the deaths of dairy animals in a survey conducted in 2000 in the US (Baker 2002). In more recent times, the incidence of HBS is thought to be increasing but additional estimates of incidence are unavailable because there is a marked seasonality to the disease, symptoms mimic common ruminant digestive diseases and a large proportion of afflicted cattle are not submitted for necropsy.

HBS was first reported in 1991, observed in five high-producing Holstein cows from one dairy in Idaho (Sockett, 2004). Symptoms included point-source sub-mucosal hematomas, each affecting 10-20 cm of the jejunum. One of the five cows exhibited a ruptured hematoma with exsanguination into the lumen of the jejunum. Common symptoms of HBS include a sudden drop in milk production, abdominal pain due to obstructed bowel and anemia (Anderson, 2002). Clinical signs of the disease are decreased feed intake, depression, decreased milk production, dehydration, abdominal distension and dark clotted blood in the feces (Dennison et al., 2002). Death comes within 48 hours from the onset of the obstructing blood clot plug.

Although Aspergillus fumigatus and Clostridium perfringens are known to be involved in the etiology of HBS (Ceci et al., 2006; Dennison et al., 2005), the syndrome is better described as being poly-microbial and multi-factorial in nature. Increased consumption of a high-energy diet seems to be the most plausible common pathway for all the risk factors that have been described (Berghaus et al., 2005).

In addition to Clostridium species causing enteric disease, other Clostridium species isolated from rumen fluid and fecal samples of dairy cows are species known to produce high levels of acetone, butanol, 1,3 propanediol and butyric acid as end products of their metabolism. These metabolic end products are known to inhibit rumen and gastrointestinal bacteria and can affect rumen and digestive function and decrease efficiency. If present in the silage these organisms can reduce nutritional value of the crop.

Due to the sporadic, acute etiology of enteric clostridial infections therapeutic treatments are not known to be highly efficacious. Therefore, prophylactic strategies such as the use of probiotics to control clostridial proliferation in the GI tract are the preferred direction for disease control. Non-toxigenic clostridial challenges can also be controlled using probiotics. In accordance with one aspect of the present invention, inventors have conducted a search for an effective probiotic capable of inhibiting a broad range of pathogenic and non-pathogenic clostridia species. Over eons, Bacillus have competed with clostridia in the soil ecosystem. Through this process, certain strains of the genus Bacillus have developed effective mechanisms for inhibiting clostridia species. In accordance with one aspect of the present invention, the inventors have isolated and identified several strains of Bacillus capable of inhibiting a broad spectrum of clostridia that impact ruminant productivity. The predominant bacteriocins produced by bacilli are a variety of functionally and structurally diverse peptides. They are often hydrophobic and cyclic with unusual amino acids and resistant to peptidases and proteases. They may be synthesized ribosomally or nonribosomally by multi-enzyme complexes, often followed by post-translational modifications. Bacillus strains often produce nonribosomally synthesized lipopeptides, fatty acids attached to small cyclic peptides. These nonribosomally synthesized peptides are structurally diverse (Luo et al., 2015a), as they are assembled from a heterogeneous group of precursors, but their synthesis by a multicarrier thiotemplate mechanism is conserved (Luo et al., 2015b).

Silage is a significant source of clostridial organisms in ruminant production systems. Silage and forages can support the growth of a variety of spoilage microorganisms, such as clostridia, bacilli, yeasts and molds that contribute to the degradation of nutrient value. Because of the many variables that prevent ideal conditions for preserving silage, lactic acid bacteria are often utilized as silage inoculates to promote proper fermentation and optimal preservation of silage. Lactic acid bacteria grow quickly in anaerobic conditions and become the dominant microorganisms present in the crop, and lower the pH through the production of lactic acid. Although coliforms and molds are inhibited by lowering the pH to less than pH 5, clostridia are more difficult to control with low pH as they can survive even at pHs less than 5.0. Therefore, traditional lactic acid bacteria silage inoculates are not completely effective at controlling clostridia in silage.

Controlling clostridia organisms in silage is important to prevent the detrimental effects these bacteria have on silage quality and ruminant performance. Clostridia activity in silage is undesirable due to the reduced intake observed in cattle when the clostridia activity is present and because of the reduced nutritional quality of the silage that results from clostridia fermentation. The fermentation of lactic acid to butyric acid by the butyrate producing clostridia results in approximately 50% loss in dry matter and 18% loss in gross energy from the silage feedstuff (McDonald et al., 1991). Furthermore, clostridia spoilage organisms have a detrimental effect on the health of the cattle as evidenced by greater incidence of acidosis when cattle are fed clostridial silage (Seglar, 2003).

Although bacilli are considered silage spoilage organisms, some members of the Bacillus genera are known to produce antimicrobial compounds capable controlling the growth and survival of clostridia (Hong et al., 2005). Bacilli can result in accelerating the spoilage of silage following exposure to oxygen, but rarely impact fermentation of the crop under the anaerobic conditions of the silo (Muck, 2010). Therefore, Bacillus organisms could be used at the time of ensiling to control the growth of clostridia spoilage organisms. Bacillus strains identified in one embodiment of the present invention, produce multiple compounds with inhibitory activity against a wide variety of clostridia.

Bacillus strains impact the overall ecology of the rumen and intestinal tract by inhibiting the clostridia that produce non-nutritional end-products such as acetone and butanol, which can negatively impact rumen function. The activity of Bacillus strains reduces not only the levels, but also the overall diversity of C. perfringens and non-toxigenic clostridia.

The immunomodulatory activities attributed to Bacillus strains used as probiotics is one of many ways in which they contribute to overall health and well-being (reviewed by Hong et al., 2005). Bacillus spores have been reported to pass into the intestinal Peyer's patches and mesenteric lymph nodes following oral administration, and to be phagocytosed by macrophages in vitro studies (Duc et al., 2003, 2004). The oral administration of B. subtilis induced production of the cytokine, interferon-X, by mononuclear cells in the blood (Kosaka et al., 1998), indicating Bacillus elicit a systemic effect as well as a local effect at the intestinal level. Furthermore, an increase in the messenger RNA expression of toll-like receptor (TLR)-2 and TLR-4 in the intestinal jejunum and ileum was observed following administration to broiler chickens as well as members of the downstream TLR signaling pathway. MyD88, TRAF6 and NF-κB (Rajput et al., 2017).

Bacillus strains have a number of activities that make them efficacious as direct-fed microbials including the production of extracellular enzymes, antimicrobials and immune modulating molecules. In addition, Bacillus form endospores that make them stable in feed and other feed components. These spores are heat resistant and thus will survive normal feed pelleting processes. The spores are recalcitrant to drying and mineral salts making them stable in vitamin and trace mineral premixes.

Both in vitro data and in vivo trials indicate the effectiveness of these Bacillus strains in inhibiting clostridia, such as C. perfringens, thereby decreasing the disease-burden in commercial dairy operations. These same trials have demonstrated the effectiveness of the Bacillus strains to inhibit non-toxigenic clostridia capable of producing inhibitory metabolites. These strains are now available as probiotics for improving dairy productivity and reducing digestive disease in cattle.

BRIEF SUMMARY

The inventors have developed a direct fed microbial composition comprising an isolated Bacillus strain for use in reducing a Clostridium in a digestive system of a ruminant having ingested an effective amount of said direct fed microbial composition, wherein the isolated Bacillus strain selected from the group consisting of: Bacillus subtilis 1104, Bacillus subtilis 1781, Bacillus subtilis 747, Bacillus subtilis 1541, Bacillus subtilis 1999, and Bacillus subtilis 2018.

In one aspect of the invention, a direct fed microbial composition may inhibit a clostridia selected from a group consisting of: Clostridium perfringens, Clostridium bifermentans, Clostridium beijerinckii, Clostridium butyricum, Clostridium tertium, and Clostridium sordellii.

In one aspect of the invention, wherein the Clostridium is Clostridium bifermentans, the isolated Bacillus strain may inhibit the production of 1, 3-propanediol in the digestive system of the ruminant having ingested an effective amount of said direct fed microbial composition.

In one aspect of the invention, wherein the Clostridium is Clostridium beijerinckii, the isolated Bacillus strain may inhibit the production of butanol in the digestive system of the ruminant having ingested an effective amount of said direct fed microbial composition.

In one aspect of the invention, wherein the Clostridium is Clostridium beijerinckii, the isolated Bacillus strain may inhibit the production of acetone in the digestive system of the ruminant having ingested an effective amount of said direct fed microbial composition.

In one aspect of the invention, wherein the Clostridium is Clostridium butyricum, the isolated Bacillus strain may inhibit the production of butyrate in the digestive system of the ruminant having ingested an effective amount of said direct fed microbial composition.

In one aspect of the invention, wherein the Clostridium is Clostridium perfringens, the isolated Bacillus strain may reduce the occurrence of a digestive disorder such as hemorrhagic bowel syndrome in the ruminant having ingested an effective amount of said direct fed microbial composition.

In one aspect of the invention, the direct fed microbial composition may further comprise a cryoprotectant disposed about the isolated Bacillus strain, and wherein said isolated Bacillus strain is a biologically pure, powdered lyophilized strain.

In one aspect of the invention, the direct fed microbial composition may be a biologically pure, powdered lyophilized Bacillus strain that comprises Bacillus spores.

In one aspect of the invention, the direct fed microbial composition may further comprising a carrier.

In one aspect of the invention, the effective amount of the direct fed microbial composition ingested by the ruminant per day may comprise a concentration of the isolated Bacillus strain of between about 2×10⁸ CFU/ruminant and about 2.0×10¹⁰ CFU/ruminant.

In one aspect of the invention, the effective amount of the direct fed microbial composition ingested by the ruminant per day may comprise a concentration of the isolated Bacillus strain of about 2×10⁹ CFU/ruminant.

In another aspect of the invention, a method of improving ruminant performance is provided, comprising the steps of introducing into the digestive system of one or more ruminants an effective amount of the direct fed microbial composition according to claim 1, and providing at least one benefit chosen from: (1) inhibiting a pathogen chosen from at least one of Clostridium perfringens, Clostridium bifermentans, Clostridium beijerinckii, Clostridium butyricum, Clostridium tertium, and Clostridium sordellii. in the one or more ruminants; (2) decreasing a mortality rate of the one or more ruminants; (3) improving the feed efficiency of the one or more ruminants; (4) reducing the occurrence of Hemorrhagic bowel syndrome in the one or more ruminants; (5) improve rumen fermentation in the one or more ruminants; (6) improve milk production in the one or more ruminants; and, (7) modulating immune responses of inflammatory cytokines in systemic and intestinal immune cells in the one or more ruminants.

In another aspect of the invention, the method of administering the direct fed microbial composition may provide the benefit of decreasing diversity of Clostridium perfringens strains in the one or more ruminants.

In another aspect of the invention, the method of administering the direct fed microbial composition may provide the benefit of decreasing diversity of non-toxigenic clostridial strains in the one or more ruminants.

In another aspect of the invention, the method of administering the direct fed microbial composition may provide the benefit of increasing average energy corrected milk production in the one or more ruminants when the one or more ruminants are dairy cows.

In another aspect of the invention, the method of administering the direct fed microbial composition may provide the benefit of decreasing a digestive system related mortality rate of the one or more ruminants during a period of direct fed microbial administration.

In another aspect of the invention, the method of administering the direct fed microbial composition may include adding the direct fed microbial composition to a ruminant feed.

In another aspect of the invention, the inventors have developed a cryoprotectant disposed about a powdered lyophilized isolated Bacillus strain of spores chosen from at least one of: Bacillus subtilis 1104, Bacillus subtilis 1781, Bacillus subtilis 747, Bacillus subtilis 1541, Bacillus subtilis 1999, and Bacillus subtilis 2018; and a carrier, wherein the composition may inhibit at least one pathogen selected from: Clostridium perfringens, Clostridium bifermentans, Clostridium beijerinckii, and Clostridium butyricum in a digestion system of a ruminant having ingested an effective amount of said direct fed microbial composition, and wherein the effective amount of said direct fed microbial composition may comprise a concentration of the isolated Bacillus strain of between about 2×10⁸ CFU/ruminant/day and about 2.0×10¹⁰ CFU/ruminant/day.

In another aspect of the invention, the inventors have developed a composition for reducing a Clostridium comprising an effective amount of a biologically pure culture of a Bacillus strain selected from the group consisting of Bacillus 1104, Bacillus 1781, Bacillus 747, Bacillus 1541, Bacillus 1999, and Bacillus 2018.

In another aspect of the invention, the Clostridium inhibited is selected from a group consisting of Clostridium perfringens, Clostridium bifermentans, Clostridium beijerinckii, and Clostridium butyricum, Clostridium tertium, and Clostridium sordellii.

In another aspect of the invention, the composition also comprising a cryoprotectant disposed about the isolated Bacillus strain, and said isolated Bacillus strain is a powdered lyophilized strain.

In another aspect of the invention, the composition includes biologically pure, powdered lyophilized Bacillus strain is in the form of Bacillus spores.

In another aspect of the invention, the composition may be used as a direct fed microbial to control the clostridia in a digestive system of a ruminant having ingested an effective amount of said direct fed microbial.

In another aspect of the invention, the effective amount of the direct fed microbial ingested by the ruminant per day comprises a concentration of the isolated Bacillus strain of between about 2×10⁸ CFU/ruminant and about 2.0×10¹⁰ CFU/ruminant.

In another aspect of the invention, the effective amount of said direct fed microbial ingested by the ruminant per day comprises a concentration of the isolated Bacillus strain of about 2×10⁹ CFU/ruminant.

In another aspect of the invention, the composition may be used as a silage control microbial to inhibit the growth of Clostridium in a volume of silage comprising an effective amount of said composition mixed with a volume of a fodder that yields said silage.

In another aspect of the invention, the biologically pure culture of the Bacillus strain inhibits growth of a pathogenic microorganism selected from the group consisting of E. coli, Clostridium perfringens, Clostridium bifermentans, Clostridium beijerinckii, Clostridium butyricum, Clostridium tertium, Clostridium sordellii, coliforms, yeasts, and molds.

In another aspect of the invention, the biologically pure culture of the Bacillus strain increases concentration of lactic acid and acetic acid in the silage.

In another aspect of the invention, the biologically pure culture of the Bacillus strain reduces spoilage of the silage.

In another aspect of the invention, a method for reducing growth of pathogenic microorganisms in silage comprising mixing a volume of a fodder with an effective amount of the composition to reduce growth of the pathogenic microorganism is provided.

In another aspect of the invention, a Bacillus strain is selected from the group consisting of Bacillus 1104, Bacillus 1781, Bacillus 747, Bacillus 1541, Bacillus 1999, and Bacillus 2018 for use in a direct fed microbial to control a Clostridium in a digestive systems of a ruminant.

In another aspect of the invention, a Bacillus strain is selected from the group consisting of Bacillus 1104, Bacillus 1781, Bacillus 747, Bacillus 1541, Bacillus 1999, and Bacillus 2018 for use in manufacture of a direct fed microbial to control a Clostridium in a digestive systems of a ruminant.

In another aspect of the invention, a Bacillus strain is selected from the group consisting of Bacillus 1104, Bacillus 1781, Bacillus 747, Bacillus 1541, Bacillus 1999, and Bacillus 2018 for use in a silage control microbial to inhibit the growth of a Clostridium in silage.

In another aspect of the invention, a Bacillus strain is selected from the group consisting of Bacillus 1104, Bacillus 1781, Bacillus 747, Bacillus 1541, Bacillus 1999, and Bacillus 2018 for use in manufacture of a silage control microbial to inhibit the growth of a Clostridium in silage.

Other objects, features and advantages of the present invention will become apparent after review of the specification, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a pie chart showing proportions of Clostridium perfringens and non-toxigenic clostridia isolated from feed samples in accordance with one embodiment of the present invention, pursuant to Example 1;

FIG. 2 is a pie chart showing the major non-toxigenic clostridia identified in feed samples (n=345) in accordance with one embodiment of the present invention, pursuant to Example 1;

FIG. 3 is a dendrogram displaying the differences between feed clostridia isolates based on genetic fingerprints generated by RAPD PCR in accordance with one embodiment of the present invention, pursuant to Example 1;

FIG. 4 is a line graph showing measurements of pH versus time for six mini-silo inoculant treatments in accordance with one embodiment of the present invention, pursuant to Example 2;

FIG. 5 is a line graph showing change in counts of Bacillus over time for six mini-silo inoculant treatments in accordance with one embodiment of the present invention, pursuant to Example 2;

FIG. 6 is a line graph showing lactic acid bacteria (LAB) population change over time for six mini-silo inoculant treatments in accordance with one embodiment of the present invention, pursuant to Example 2;

FIG. 7 is a line graph showing change in yeast population over time for six mini-silo inoculant treatments in accordance with one embodiment of the present invention, pursuant to Example 2;

FIG. 8 is a line graph showing change in mold population over time for the six silage inoculant treatments in accordance with one embodiment of the present invention, pursuant to Example 2;

FIG. 9 is a line graph showing change in Clostridia population over time for the six silage inoculant treatments in accordance with one embodiment of the present invention, pursuant to Example 2;

FIG. 10 is a line graph showing change in lactic acid concentration over time for the six silage inoculant treatments on an as-sampled basis in accordance with one embodiment of the present invention, pursuant to Example 2;

FIG. 11 is a line graph showing change in acetic acid concentration over time for the six silage inoculant treatments on an as-sampled basis in accordance with one embodiment of the present invention, pursuant to Example 2;

FIG. 12 is a line graph showing pH measurements over time for the four silage inoculant treatments on an as-sampled basis in accordance with one embodiment of the present invention, pursuant to Example 3;

FIG. 13 is a line graph showing E. coli population change over time for the four silage inoculant treatments on an as-sampled basis in accordance with one embodiment of the present invention, pursuant to Example 3;

FIG. 14 is a line graph showing Coliform population change over time for the four silage inoculant treatments on an as-sampled basis in accordance with one embodiment of the present invention, pursuant to Example 3;

FIG. 15 is a line graph showing Bacillus population change over time for the four silage inoculant treatments on an as-sampled basis in accordance with one embodiment of the present invention, pursuant to Example 3;

FIG. 16 is a line graph showing Lactic acid bacteria (LAB) population change over time for the four silage inoculant treatments on an as-sampled basis in accordance with one embodiment of the present invention, pursuant to Example 3;

FIG. 17 is a line graph showing yeast population change over time for the four silage inoculant treatments on an as-sampled basis in accordance with one embodiment of the present invention, pursuant to Example 3;

FIG. 18 is a line graph showing mold population change over time for the four silage inoculant treatments on an as-sampled basis in accordance with one embodiment of the present invention, pursuant to Example 3;

FIG. 19 is a line graph showing clostridia population change over time for the four silage inoculant treatments on an as-sampled basis in accordance with one embodiment of the present invention, pursuant to Example 3;

FIG. 20 is a line graph showing lactic acid level over time for the four silage inoculant treatments on an as-sampled basis in accordance with one embodiment of the present invention, pursuant to Example 3;

FIG. 21 is a line graph showing acetic acid level change over time for the four silage inoculant treatments on an as-sampled basis in accordance with one embodiment of the present invention, pursuant to Example 3;

FIG. 22 is a line graph showing pH measurements over time comparing on-farm application of a silage inoculant containing Bacillus and an untreated control in accordance with one embodiment of the present invention, pursuant to Example 4;

FIG. 23 is a line graph showing E. coli counts (CFU/g) over time comparing on-farm application of a silage inoculant containing Bacillus and an untreated control in accordance with one embodiment of the present invention, pursuant to Example 4;

FIG. 24 is a line graph showing coliform counts (CFU/g) over time comparing on-farm application of a silage inoculant containing Bacillus and an untreated control in accordance with one embodiment of the present invention, pursuant to Example 4;

FIG. 25 is a line graph showing Bacillus counts (CFU/g) over time comparing on-farm application of a silage inoculant containing Bacillus and an untreated control in accordance with one embodiment of the present invention, pursuant to Example 4;

FIG. 26 is a line graph showing lactic acid bacteria (LAB) counts (CFU/g) over time comparing on-farm application of a silage inoculant containing Bacillus and an untreated control in accordance with one embodiment of the present invention, pursuant to Example 4;

FIG. 27 is a line graph showing yeast counts (CFU/g) over time comparing on-farm application of a silage inoculant containing Bacillus and an untreated control in accordance with one embodiment of the present invention, pursuant to Example 4;

FIG. 28 is a line graph showing mold counts (CFU/g) over time comparing on-farm application of a silage inoculant containing Bacillus and an untreated control in accordance with one embodiment of the present invention, pursuant to Example 4;

FIG. 29 is a line graph showing clostridia counts (CFU/g) over time comparing on-farm application of a silage inoculant containing Bacillus and an untreated control in accordance with one embodiment of the present invention, pursuant to Example 4;

FIG. 30 is a line graph showing lactic acid concentration over time comparing on-farm application of a silage inoculant containing Bacillus and an untreated control in accordance with one embodiment of the present invention, pursuant to Example 4;

FIG. 31 is a line graph showing acetic acid concentration over time comparing on-farm application of a silage inoculant containing Bacillus and an untreated control in accordance with one embodiment of the present invention, pursuant to Example 4;

FIG. 32 is a chart showing enumeration results of all clostridia (no differentiation of Clostridium sp.) by individual fecal sample for cows and calves in Wisconsin in accordance with one embodiment of the present invention, pursuant to Example 5;

FIG. 33 is a chart showing enumeration results of total clostridia (no differentiation of Clostridium sp.) by individual farm calf fecal samples for each farm sampled in Wisconsin in accordance with one embodiment of the present invention, pursuant to Example 5;

FIG. 34 is a chart showing enumeration results of total clostridia (no differentiation of Clostridium sp.) by individual farm cow fecal samples for each farm sampled from Wisconsin in accordance with one embodiment of the present invention, pursuant to Example 5;

FIG. 35 is a chart showing calculated counts of C. perfringens by fecal sample, from fecal samples collected in Wisconsin. C. perfringens counts were estimated by multiplying each sample's total clostridia count by the percent that were confirmed to be C. perfringens in accordance with one embodiment of the present invention, pursuant to Example 5;

FIG. 36 is a chart showing calculated counts of C. perfringens by individual calf fecal sample from Wisconsin. C. perfringens counts were estimated by multiplying each sample's clostridia count by the percent that were confirmed to be C. perfringens. Results are reported for each farm in accordance with one embodiment of the present invention, pursuant to Example 5;

FIG. 37 is a chart showing calculated counts of C. perfringens by individual cow fecal sample collected from Wisconsin. C. perfringens counts were estimated by multiplying each sample's clostridia count by the percent that were confirmed to be C. perfringens. Results are reported for each farm in accordance with one embodiment of the present invention, pursuant to Example 5;

FIG. 38 is a dendrogram showing Clostridium perfringens (n=1,522) isolated from Wisconsin, with a cluster cutoff at 75% similarity wherein information about each isolate (n=1,522) is listed on the right: farm, ID number, Alpha, Beta, Epsilon and Iota toxin results, Toxin type, Source and State; and representatives were selected based on dendrogram clusters for screening in bacteriocin inhibition assays in accordance with one embodiment of the present invention, pursuant to Example 5;

FIG. 39 is a dendrogram of non-toxigenic clostridia (n=183) displaying similarities between 16S DNA sequences from each isolate from Wisconsin; wherein information about each isolate is listed from left to right, including: farm, ID number or Type strain identification, identification, source of isolate in accordance with one embodiment of the present invention, pursuant to Example 5;

FIG. 40 is a pie graph showing major non-toxigenic clostridia (n=183) species compared to all other non-toxigenic types in accordance with one embodiment of the present invention, pursuant to Example 5;

FIG. 41 is a chart showing enumeration results of all clostridia (no differentiation of Clostridium sp.) by individual fecal sample for cows and calves in Texas in accordance with one embodiment of the present invention, pursuant to Example 6;

FIG. 42 is a chart showing enumeration results of total clostridia (no differentiation of Clostridium sp.) by individual farm calf fecal samples for each farm sampled in Texas in accordance with one embodiment of the present invention, pursuant to Example 6;

FIG. 43 is a chart showing enumeration results of total clostridia (no differentiation of Clostridium sp.) by individual farm cow fecal samples for each farm sampled from Texas in accordance with one embodiment of the present invention, pursuant to Example 6;

FIG. 44 is a chart showing calculated counts of C. perfringens by fecal sample, from fecal samples collected in Texas, where C. perfringens counts were estimated by multiplying each sample's total clostridia count by the percent that were confirmed to be C. perfringens in accordance with one embodiment of the present invention, pursuant to Example 6;

FIG. 45 is a chart showing calculated counts per farm of C. perfringens by individual calf fecal samples from Texas, where C. perfringens counts were estimated by multiplying each sample's clostridia count by the percent that were confirmed to be C. perfringens in accordance with one embodiment of the present invention, pursuant to Example 6;

FIG. 46 is a chart showing calculated counts per farm of C. perfringens by individual cow fecal sample collected from Texas, where C. perfringens counts were estimated by multiplying each sample's clostridia count by the percent that were confirmed to be C. perfringens in accordance with one embodiment of the present invention, pursuant to Example 6;

FIG. 47 is a pie graph showing the major species of non-toxigenic clostridia (n=215) isolated from Texas samples compared to all other non-toxigenic types in accordance with one embodiment of the present invention, pursuant to Example 6;

FIG. 48 is a chart showing enumeration results of all clostridia (no differentiation of Clostridium sp.) by individual fecal sample for cows and calves from the Upper Midwest region in accordance with one embodiment of the present invention, pursuant to Example 7;

FIG. 49 is a chart showing enumeration results of total clostridia (no differentiation of Clostridium sp.) by individual farm calf fecal samples for each farm sampled from the Upper Midwest region in accordance with one embodiment of the present invention, pursuant to Example 7;

FIG. 50 is a chart showing enumeration results of total clostridia (no differentiation of Clostridium sp.) by individual farm cow fecal samples for each farm sampled from the Upper Midwest region in accordance with one embodiment of the present invention, pursuant to Example 7;

FIG. 51 is a chart showing calculated counts of C. perfringens by fecal sample, from fecal samples collected from the Upper Midwest region, where C. perfringens counts were estimated by multiplying each sample's total clostridia count by the percent that were confirmed to be C. perfringens in accordance with one embodiment of the present invention, pursuant to Example 7;

FIG. 52 is a chart showing calculated counts of C. perfringens for each farm by individual calf fecal sample from the Upper Midwest region, where C. perfringens counts were estimated by multiplying each sample's clostridia count by the percent that were confirmed to be C. perfringens in accordance with one embodiment of the present invention, pursuant to Example 7;

FIG. 53 is a chart showing calculated counts of C. perfringens for each farm by individual cow fecal sample collected from the Upper Midwest region, where C. perfringens counts were estimated by multiplying each sample's clostridia count by the percent that were confirmed to be C. perfringens in accordance with one embodiment of the present invention, pursuant to Example 7;

FIG. 54 is a pie graph showing Pie graph of non-toxigenic clostridia (n=218), showing the two major identification types (the C. bifermentans group and the C. beijerinckii group) compared to all other non-toxigenic types isolated from the Upper Midwest region in accordance with one embodiment of the present invention, pursuant to Example 7;

FIG. 55 is a chart showing enumeration results of all clostridia (no differentiation of Clostridium sp.) by individual fecal sample for cows and calves from the Great Lakes region in accordance with one embodiment of the present invention, pursuant to Example 7;

FIG. 56 is a chart showing enumeration results of total clostridia (no differentiation of Clostridium sp.) by individual farm calf fecal samples for each farm sampled from the Great Lakes region in accordance with one embodiment of the present invention, pursuant to Example 8;

FIG. 57 is a chart showing enumeration results of total clostridia (no differentiation of Clostridium sp.) by individual farm cow fecal samples for each farm sampled from the Great Lakes region in accordance with one embodiment of the present invention, pursuant to Example 8;

FIG. 58 is a chart showing calculated counts of C. perfringens by fecal sample, from fecal samples collected from the Great Lakes region, where C. perfringens counts were estimated by multiplying each sample's total clostridia count by the percent that were confirmed to be C. perfringens in accordance with one embodiment of the present invention, pursuant to Example 8;

FIG. 59 is a chart showing calculated counts of C. perfringens for each farm by individual calf fecal sample from the Great Lakes region, where C. perfringens counts were estimated by multiplying each sample's clostridia count by the percent that were confirmed to be C. perfringens in accordance with one embodiment of the present invention, pursuant to Example 8;

FIG. 60 is a chart showing calculated counts of C. perfringens for each farm by individual cow fecal sample collected from the Great Lakes region. C. perfringens counts were estimated by multiplying each sample's clostridia count by the percent that were confirmed to be C. perfringens in accordance with one embodiment of the present invention, pursuant to Example 8;

FIG. 61 is a pie graph showing non-toxigenic clostridia (n=190), including the major identification type (the C. bifermentans group) compared to all other non-toxigenic types isolated from the Great Lakes region in accordance with one embodiment of the present invention, pursuant to Example 8;

FIG. 62 is a chart showing enumeration results of all clostridia (no differentiation of Clostridium sp.) by individual fecal sample for cows and calves from the Northeast region in accordance with one embodiment of the present invention, pursuant to Example 9;

FIG. 63 is a chart showing enumeration results of total clostridia (no differentiation of Clostridium sp.) by individual farm calf fecal samples for each farm sampled from the Northeast region in accordance with one embodiment of the present invention, pursuant to Example 9;

FIG. 64 is a chart showing enumeration results of total clostridia (no differentiation of Clostridium sp.) by individual farm cow fecal samples for each farm sampled from the Northeast region in accordance with one embodiment of the present invention, pursuant to Example 9;

FIG. 65 is a chart showing calculated counts of C. perfringens by fecal sample, from fecal samples collected from the Northeast region, where C. perfringens counts were estimated by multiplying each sample's total clostridia count by the percent that were confirmed to be C. perfringens in accordance with one embodiment of the present invention, pursuant to Example 9;

FIG. 66 is a chart showing calculated counts of C. perfringens for each farm by individual calf fecal sample from the Northeast region, where C. perfringens counts were estimated by multiplying each sample's clostridia count by the percent that were confirmed to be C. perfringens in accordance with one embodiment of the present invention, pursuant to Example 9;

FIG. 67 is a chart showing calculated counts of C. perfringens for each farm by individual cow fecal sample collected from the Northeast region, wherein C. perfringens counts were estimated by multiplying each sample's clostridia count by the percent that were confirmed to be C. perfringens in accordance with one embodiment of the present invention, pursuant to Example 9;

FIG. 68 is a pie graph showing non-toxigenic clostridia (n=181), including the two major identification types (the C. bifermentans group and the C. beijerinckii group) compared to all other non-toxigenic types isolated from the Northeast region in accordance with one embodiment of the present invention, pursuant to Example 9;

FIG. 69 is a chart showing enumeration results of all clostridia (no differentiation of Clostridium sp.) by individual fecal sample for cows and calves from the Mid-Atlantic region in accordance with one embodiment of the present invention, pursuant to Example 10;

FIG. 70 is a chart showing enumeration results of total clostridia (no differentiation of Clostridium sp.) by individual farm calf fecal samples for each farm sampled from the Mid-Atlantic region in accordance with one embodiment of the present invention, pursuant to Example 10;

FIG. 71 is a chart showing enumeration results of total clostridia (no differentiation of Clostridium sp.) by individual farm cow fecal samples for each farm sampled from the Mid-Atlantic region in accordance with one embodiment of the present invention, pursuant to Example 10;

FIG. 72 is a chart showing calculated counts of C. perfringens by fecal sample, from fecal samples collected from the Mid-Atlantic region, where C. perfringens counts were estimated by multiplying each sample's total clostridia count by the percent that were confirmed to be C. perfringens in accordance with one embodiment of the present invention, pursuant to Example 10;

FIG. 73 is a chart showing calculated counts of C. perfringens for each farm by individual calf fecal sample from the Mid-Atlantic region, where C. perfringens counts were estimated by multiplying each sample's clostridia count by the percent that were confirmed to be C. perfringens in accordance with one embodiment of the present invention, pursuant to Example 10;

FIG. 74 is a chart showing calculated counts of C. perfringens for each farm by individual cow fecal sample collected from the Mid-Atlantic region, where C. perfringens counts were estimated by multiplying each sample's clostridia count by the percent that were confirmed to be C. perfringens in accordance with one embodiment of the present invention, pursuant to Example 10;

FIG. 75 is a pie graph showing non-toxigenic clostridia (n=151), including the major identification type, the C. bifermentans group, compared to all other non-toxigenic types isolated from the Mid-Atlantic region in accordance with one embodiment of the present invention, pursuant to Example 10;

FIG. 76 is a chart showing enumeration results of all clostridia (no differentiation of Clostridium sp.) by individual fecal sample for cows and calves from the 1-29 Corridor region in accordance with one embodiment of the present invention, pursuant to Example 11;

FIG. 77 is a chart showing enumeration results of total clostridia (no differentiation of Clostridium sp.) by individual farm calf fecal samples for each farm sampled from the 1-29 Corridor region in accordance with one embodiment of the present invention, pursuant to Example 11;

FIG. 78 is a chart showing enumeration results of total clostridia (no differentiation of Clostridium sp.) by individual farm cow fecal samples for each farm sampled from the 1-29 Corridor region in accordance with one embodiment of the present invention, pursuant to Example 11;

FIG. 79 is a chart showing calculated counts of C. perfringens by fecal sample, from fecal samples collected from the 1-29 Corridor region, where C. perfringens counts were estimated by multiplying each sample's total clostridia count by the percent that were confirmed to be C. perfringens in accordance with one embodiment of the present invention, pursuant to Example 11;

FIG. 80 is a chart showing calculated counts of C. perfringens for each farm by individual calf fecal sample from the 1-29 Corridor region, where C. perfringens counts were estimated by multiplying each sample's clostridia count by the percent that were confirmed to be C. perfringens in accordance with one embodiment of the present invention, pursuant to Example 11;

FIG. 81 is a chart showing calculated counts of C. perfringens for each farm by individual cow fecal sample collected from the 1-29 Corridor region, where C. perfringens counts were estimated by multiplying each sample's clostridia count by the percent that were confirmed to be C. perfringens in accordance with one embodiment of the present invention, pursuant to Example 11;

FIG. 82 is a pie graph showing non-toxigenic clostridia (n=399), including the two major identification types (the C. bifermentans group and the C. beijerinckii group) compared to all other non-toxigenic types isolated from the 1-29 Corridor region in accordance with one embodiment of the present invention, pursuant to Example 11;

FIG. 83 is a chart showing enumeration results of total clostridia by individual fecal sample for all time points, including those of Example 6, where time points that are significantly different from the pretreatment samples are indicated by asterisks over the mean error bars, in accordance with one embodiment of the present invention, pursuant to Example 12;

FIG. 84 is a chart showing calculated counts of Clostridium perfringens by individual fecal sample for all time points, including those of Example 6 in accordance with one embodiment of the present invention, pursuant to Example 12;

FIG. 85 is a bar graph showing changes in the proportions of non-toxigenic Clostridium species over time at Farm ALJ in Texas in accordance with one embodiment of the present invention, pursuant to Example 12;

FIG. 86 is a chart showing enumeration results of total clostridia by individual fecal sample for all sampling points for Wisconsin Farm E in accordance with one embodiment of the present invention, pursuant to Example 13;

FIG. 87 is a chart showing calculated counts of Clostridium perfringens by individual fecal sample over time from Wisconsin Farm E in accordance with one embodiment of the present invention, pursuant to Example 13;

FIG. 88 is a graph showing proportions of non-toxigenic Clostridium species over time from Wisconsin Farm E in accordance with one embodiment of the present invention, pursuant to Example 13;

FIG. 89 is a chart showing enumeration results from Farm BS of total clostridia by individual fecal sample for all time points, including those of Example 6, where Time points that are significantly different from the Pretreatment samples are indicated by asterisks over the mean error bars in accordance with one embodiment of the present invention, pursuant to Example 14;

FIG. 90 is a chart showing calculated counts from Farm BS of Clostridium perfringens by individual fecal sample for all time points, including those of Example 6 in accordance with one embodiment of the present invention, pursuant to Example 14;

FIG. 91 is a graph showing proportions of non-toxigenic Clostridium species from the pretreatment compared to 107 days treated sampling points from Texas Farm BS in accordance with one embodiment of the present invention, pursuant to Example 14;

FIG. 92 is a graph showing fold-change in inflammatory cytokine (MIP-2 and TNF-α) gene expression elicited by Bacillus strain 747 with and without LPS, in accordance with one embodiment of the present invention, pursuant to Example 15;

FIG. 93 is a graph showing fold-change in inflammatory cytokine (MIP-2 and TNF-α) gene expression elicited by Bacillus strain 1781 with and without LPS, in accordance with one embodiment of the present invention, pursuant to Example 15;

FIG. 94 is a graph showing fold-change in inflammatory cytokine (MIP-2 and TNF-α) gene expression elicited by Bacillus strain 1104 with and without LPS, in accordance with one embodiment of the present invention, pursuant to Example 15;

FIG. 95 is a graph showing fold-change in inflammatory cytokine (MIP-2 and TNF-α) gene expression elicited by Bacillus strain 1541 with and without LPS, in accordance with one embodiment of the present invention, pursuant to Example 15;

FIG. 96 is a graph showing fold-change in inflammatory cytokine (MIP-2 and TNF-α) gene expression elicited by Bacillus strain 2018 with and without LPS, in accordance with one embodiment of the present invention, pursuant to Example 15;

FIG. 97 is a graph showing fold-change in inflammatory cytokine (MIP-2 and TNF-α) gene expression elicited by Bacillus strains 747 and 1781 with and without LPS, in accordance with one embodiment of the present invention, pursuant to Example 15;

FIG. 98 is a graph showing plasma haptoglobin concentrations of calves 14 days following the initiation of milk replacer treatments, in accordance with one embodiment of the present invention, pursuant to Example 16;

FIG. 99 is a chart showing enumeration results from Wisconsin Farm WB of total clostridia by individual fecal sample for all time points, including those of Example 5, where time points that are significantly different from the Pretreatment samples are indicated by asterisks over the mean error bars in accordance with one embodiment of the present invention, pursuant to Example 18;

FIG. 100 is a chart showing calculated counts of Clostridium perfringens from Wisconsin Farm WB by individual fecal sample for all time points, including those of Example 5 in accordance with one embodiment of the present invention, pursuant to Example 18; and

FIG. 101 is a chart showing proportions of non-toxigenic Clostridium species over time from Wisconsin Farm WB in accordance with one embodiment of the present invention, pursuant to Example 18.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range.

It is noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

The numerical ranges in this disclosure are approximate, and thus may include values outside of the range unless otherwise indicated. Numerical ranges include all values from and including the lower and the upper values, in increments of one unit, provided that there is a separation of at least two units between any lower value and any higher value. For ranges containing values which are less than one or containing fractional numbers greater than one (e.g., 1.1, 1.5, etc.), one unit is considered to be 0.0001, 0.001, 0.01 or 0.1, as appropriate. For ranges containing single digit numbers less than ten (e.g., 1 to 5), one unit is typically considered to be 0.1. These are only examples of what is specifically intended, and all possible combinations of numerical values between the lowest value and the highest value enumerated, are to be considered to be expressly stated in this disclosure. Numerical ranges are provided within this disclosure for, among other things, relative amounts of components in a mixture, and various temperature and other parameter ranges recited in the methods.

As used herein, “administer” is meant the action of introducing the strain or a composition to an environment.

As used herein, the term “animal” includes but is not limited to human, mammal, amphibian, bird, reptile, pigs, cows, cattle, goats, horses, sheep, poultry, and other animals kept or raised on a farm or ranch, sheep, big-horn sheep, buffalo, antelope, oxen, donkey, mule, deer, elk, caribou, water buffalo, camel, llama, alpaca, rabbit, mouse, rat, guinea pig, hamster, ferret, dog, cat, and other pets, primate, monkey, ape, and gorilla. In some embodiments, the animals are ruminants, including but not limited to cattle, sheep, goats, etc.

As used herein, “animal performance” may be determined by the feed efficiency and/or weight gain of the animal and/or by the feed conversion ratio and/or by the digestibility of a nutrient in a feed (e.g. amino acid digestibility) and/or digestible energy or metabolizable energy in a feed and/or by nitrogen retention and/or by animals ability to avoid the negative effects of necrotic enteritis and/or by the immune response of the subject.

By “at least one strain,” is meant a single strain but also mixtures of strains comprising at least two strains of bacteria. By “a mixture of at least two strains,” is meant a mixture of two, three, four, five, six or even more strains. In some embodiments of a mixture of strains, the proportions can vary from 1% to 99%. When a mixture comprises more than two strains, the strains can be present in substantially equal proportions in the mixture or in different proportions.

As used herein, a “biologically pure strain” refers to a strain containing no other bacterial strains in quantities sufficient to interfere with replication of the strain or to be detectable by normal bacteriological techniques. “Isolated” when used in connection with the organisms and cultures described herein includes not only a biologically pure strain, but also any culture of organisms that is grown or maintained other than as it is found in nature.

As used herein, Clostridium perfringens (formerly known as C. welchii, or Bacillus welchii) is a Gram-positive, rod-shaped, anaerobic, spore-forming pathogenic bacterium of the genus Clostridium.

As used herein the term “contacted” refers to the indirect or direct application of the strains, silage control microbials (“SCMs”), or composition disclosed herein to a product, including but not limited to a feed. Examples of the application methods which may be used, include, but are not limited to, treating the product in a material comprising a bacterial strain, a SCM, or a composition, direct application by mixing a bacterial strain, a SCM, or a composition with the product, spraying a bacterial strain, a SCM, or a composition onto the product surface or dipping the product into a preparation of the a bacterial strain, a SCM, or a composition.

As used herein, the term “compound feed” refers to a commercial feed in the form of a meal, a pellet, nuts, cake or a crumble. Compound feeds may be blended from various raw materials and additives. These blends are formulated according to the specific requirements of the target animal.

In one embodiment, “effective amount” refers to a quantity of SCM to reduce growth of clostridia in a feed, including but not limited to silage and fodder.

In another embodiment, “effective amount” is meant a quantity of DFM to improve performance of an animal. Improvement in performance can be measured as described herein or by other methods known in the art. An effective amount can be administered to the animal by providing ad libitum access to feed containing the DFM and exogenous enzymes. The DFM and exogenous enzymes can also be administered in one or more doses.

As used herein, “energy digestibility” means the gross energy of the feed consumed minus the gross energy of the feces or the gross energy of the feed consumed minus the gross energy of the remaining digesta on a specified segment of the gastro-intestinal tract of the animal, e.g. the ileum. Metabolizable energy as used herein refers to apparent metabolizable energy and means the gross energy of the feed consumed minus the gross energy contained in the feces, urine, and gaseous products of digestion. Energy digestibility and metabolizable energy may be measured as the difference between the intake of gross energy and the gross energy excreted in the feces or the digesta present in specified segment of the gastro-intestinal tract using the same methods to measure the digestibility of nutrients, with appropriate corrections for nitrogen excretion to calculate metabolizable energy of feed.

As used herein, the term “feed” is used synonymously herein with “feedstuff.”

As used herein, the “feedstuff” may comprise feed materials comprising maize or corn, wheat, barley, triticale, rye, rice, tapioca, sorghum, and/or any of the by-products, as well as protein rich components like soybean mean, rape seed meal, canola meal, cotton seed meal, sunflower seed mean, animal-by-product meals and mixtures thereof. More preferably, the feedstuff may comprise animal fats and/or vegetable oils. The feedstuff may also contain additional minerals such as, for example, calcium and/or additional vitamins.

As used herein, the term “feed efficiency” refers to the amount of weight gain in an animal that occurs when the animal is fed ad-libitum or a specified amount of food during a period of time.

As used herein, the term “feed conversion ratio” refers to the amount of feed fed to an animal to increase the weight of the animal by a specified amount.

By “lower feed conversion ratio” or “improved feed conversion ratio” it is meant that the use of a DFM or composition in feed results in a lower amount of feed being required to be fed to an animal to increase the weight of the animal by a specified amount compared to the amount of feed required to increase the weight of the animal by the same amount when the feed does not comprise the DFM or composition.

As used herein, the term “fodder” refers to any food that is provided to an animal (rather than the animal having to forage for it themselves). Fodder encompasses plants that have been cut. The term fodder includes hay, straw, silage, compressed and pelleted feeds, oils and mixed rations, and also sprouted grains and legumes.

As used herein, “improved animal performance” means there is increased feed efficiency, and/or increased weight gain and/or reduced feed conversion ratio and/or improved digestibility of nutrients or energy in a feed and/or by improved nitrogen retention and/or by improved ability to avoid the negative effects of necrotic enteritis and/or by an improved immune response in the subject resulting from a bacterial strain, DFM, SCM, or composition disclosed herein in comparison to a subject not fed the bacterial strain, DMF, SCM, or composition.

As used herein, “immune response” means one of the multiple ways in which bacterial strains, SCMs, DFMs or compositions disclosed herein modulate the immune system of animals, including increased antibody production, up-regulation of cell mediated immunity, up-regulation of pro-inflammatory cytokines, and augmented toll-like receptor signalling. It is understood that immuno-stimulation of the gastro intestinal tract by bacterial strains, SCMs, DFMs or compositions disclosed herein may be advantageous to protect the host against disease, and that immuno-suppression of the gastro intestinal tract may be advantageous to the host because less nutrients and energy are used to support the immune function.

As used herein, the term “livestock” refers to any farmed animal. In one embodiment, livestock is one or more of ruminants such as cattle (e.g. cows or bulls (including calves)), mono-gastric animals such as poultry (including broilers, chickens and turkeys), pigs (including piglets), birds, aquatic animals such as fish, agastric fish, gastric fish, freshwater fish such as salmon, cod, trout and carp, e.g. koi carp, marine fish such as sea bass, and crustaceans such as shrimps, mussels and scallops), horses (including race horses), sheep (including lambs).

As used herein, the term “microbial” is used interchangeably with “microorganism.”

As used herein, “nitrogen retention” means a subject's ability to retain nitrogen from the diet as body mass. A negative nitrogen balance occurs when the excretion of nitrogen exceeds the daily intake and is often seen when the muscle is being lost. A positive nitrogen balance is often associated with muscle growth, particularly in growing animals. Nitrogen retention may be measured as the difference between the intake of nitrogen and the excreted nitrogen by means of the total collection of excreta and urine during a period of time. It is understood that excreted nitrogen includes undigested protein from the feed, endogenous proteinaceous secretions, microbial protein, and urinary nitrogen.

As used herein, “nutrient digestibility” means the fraction of a nutrient that disappears from the gastro-intestinal tract or a specified segment of the gastro-intestinal tract, e.g. the small intestine. Nutrient digestibility may be measured as the difference between what is administered to the subject and what comes out in the faeces of the subject, or between what is administered to the subject and what remains in the digesta on a specified segment of the gastro intestinal tract, e.g. the ileum. Nutrient digestibility may be measured by the difference between the intake of a nutrient and the excreted nutrient by means of the total collection of excreta during a period of time; or with the use of an inert marker that is not absorbed by the animal, and allows the researcher calculating the amount of nutrient that disappeared in the entire gastro-intestinal tract or a segment of the gastro-intestinal tract. Such an inert marker may be titanium dioxide, chromic oxide or acid insoluble ash. Digestibility may be expressed as a percentage of the nutrient in the feed, or as mass units of digestible nutrient per mass units of nutrient in the feed.

As used herein, “reducing the growth of microorganism” includes but is not limited to reducing the growth of microorganisms by a percentage or range of percentages at least greater than 1%.

As used herein, “silage” refers to a fermented, high-moisture stored fodder that can be fed to cattle, sheep and other such ruminants (cud-chewing animals) or used as a biofuel feedstock for anaerobic digesters. It is fermented and stored in a process called ensilage, ensiling or silaging, and is usually made from grass crops, including maize, sorghum or other cereals, using the entire green plant (not just the grain). Silage can be made from many field crops, and special terms may be used depending on type (oatlage for oats, haylage for alfalfa—but see below for the different British use of the term haylage).

As used herein, a “variant” has at least 80% identity of genetic sequences with the disclosed strains using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis. The degree of identity of genetic sequences can vary. In some embodiments, the variant has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity of genetic sequences with the disclosed strains using RAPD-PCR analysis. Six primers that can be used for RAPD-PCR analysis include the following: Primer 1 (5′-GGTGCGGGAA-3′) (SEQ ID No. 1), PRIMER 2 (5′-GTTTCGCTCC-3′) (SEQ ID No. 2), PRIMER 3 (5′-GTAGACCCGT-3′) (SEQ ID No. 3), PRIMER 4 (5′-AAGAGCCCGT-3′) (SEQ ID No. 4), PRIMER 5 (5′-AACGCGCAAC-3′) (SEQ ID No. 5), PRIMER 6 (5′-CCCGTCAGCA-3′) (SEQ ID No. 6). RAPD analysis can be performed using Ready-to-Go™ RAPD Analysis Beads (Amersham Biosciences, Sweden), which are designed as pre-mixed, pre-dispensed reactions for performing RAPD analysis.

As used herein, the term “viable microorganism” refers to a microorganism which is metabolically active or able to differentiate.

In one embodiment, the disclosure is directed to bacterial strains, SCMs, compositions and methods for controlling clostridia growth in feedstuffs. In another embodiment, the disclosure is directed to bacterial strains, SCMs, compositions and methods for controlling clostridia growth in silage.

In one embodiment, the disclosure is directed to bacterial strains, DFMs, compositions and methods for improving performance of an animal. Certain Bacillus strains and combinations and compositions thereof can be used to increase performance measures of an animal.

I. Microbials

In one embodiment, the disclosure relates to one or more bacterial strains. In yet another embodiment, the disclosure relates to compositions comprising or consisting of or consisting essentially of one or more bacterial strains. In one embodiment, a composition may be a heterogeneous mixture, a homogeneous mixture, a powder, lyophilized, freeze-dried, or any combination thereof.

A. Silage Control Microbials

Silage control microbials (SCMs) are microorganisms that reduce spoilage of a substrate, including but not limited to feed, silage and fodder. In one embodiment, the SCM comprises a viable microorganism. In another embodiment, the SCM comprises a viable bacterium.

In one embodiment the SCM may be a spore forming bacterium and hence the term SCM may be comprised of or contain spores, e.g. bacterial spores. Therefore, in one embodiment the term “viable microorganism” as used herein may include microbial spores, such as endospores.

In another embodiment, the disclosure relates to compositions that are not comprised of or do not contain microbial spores, e.g. endospores.

In one embodiment, the SCM is a combination comprising two or more bacterial strains.

In one embodiment, the bacterium or bacteria is or are isolated. In another embodiment, the SCM is a biologically pure culture of a bacterium. In still another embodiment, the SCM is a composition that comprises at least two bacterial strains that contain no other microorganisms. In still another embodiment, the SCM is a composition that comprises at least two bacterial strains that contain no other microorganisms that are found in a native environment.

In one embodiment the SCM may be a viable or inviable microorganism that is used in isolated or semi-isolated form. The SCM may be used in combination with or without the growth medium in which it was cultured.

In one embodiment, the SCM is capable of producing colony forming units when grown on an appropriate media. The appropriate media may comprise (or consist of) a feed or a feed constituent.

In one embodiment, the SCM is incapable of producing colony forming units when grown on an appropriate media. Irrespective of whether the SCM is capable or incapable of producing colony forming units when grown on an appropriate media—the cells may be still metabolically active (e.g. even if they are unable to divide).

In one embodiment the SCM may be administered as inviable cells. In one embodiment the SCM may be administered as a viable microorganism.

In one embodiment the SCM may be selected from the following Bacillus spp: Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus pumilus. In one embodiment the SCM may be a Bacillus strain.

In one embodiment the SCM may be a Bacillus subtilis. In one embodiment the SCM may be selected from the group consisting of: Bacillus subtilis 1104.

In another embodiment, the SCM may be a Bacillus subtilis. In still another embodiment, the SCM may be Bacillus subtilis 1781. In still another embodiment, the SCM may be Bacillus subtilis 747.

In another embodiment, the SCM is a multi-strain SCM comprising Bacillus subtilis 747, 1104, 1541, 1781, 1999 and 2018.

a. Formulation of a SCM

In one embodiment, the SCM formulations contained the Bacillus inoculant 50% of each strain 1104 and 1781.

In another embodiment, the SCM is a multi-strain SCM comprising Bacillus subtilis 1104 and 1781 and LAB strains. The LAB composition comprised 30% Lp115 (Lactobacillus plantarum), 30% Pj300 (Pediococcus acidilactici), 30% P751 (P. pentosaceus), and 10% Enterococcus faecium.

In another embodiment, the inoculant target application rates per gram of silage ranged for Bacillus from about 5,000 CFU/g to about 5,000,000 CFU/g. The LAB incolulant may be applied at about 150,000 CFU/g.

b. Dosing

In one embodiment, the SCM and compositions disclosed herein may be designed for one-time application. In one embodiment, the SCM and compositions disclosed herein may be mixed with a substrate, such as silage or fodder, to prevent clostridial spoilage.

The optimum amount of the composition (and each component therein) to be used in the combination may depend on the product to be treated and/or the method of contacting the product with the composition and/or the intended use for the same. The amount of SCM should be a sufficient amount to be effective and to remain sufficiently effective in reducing spoilage of a substrate.

B. Direct Fed Microbials

Direct fed microbials (DFMs) are microorganisms that improve performance of an animal. In one embodiment, the DFM comprises a viable microorganism. In another embodiment, the DFM comprises a viable bacterium.

In one embodiment the DFM may be a spore forming bacterium and hence the term DFM may be comprised of or contain spores, e.g. bacterial spores. Therefore, in one embodiment the term “viable microorganism” as used herein may include microbial spores, such as endospores or conidia.

In another embodiment, the disclosure relates to compositions that are not comprised of or do not contain microbial spores, e.g. endospores.

In one embodiment, the DFM is a combination comprising two or more bacterial strains.

In one embodiment, the bacterium or bacteria is or are isolated. In another embodiment, the DFM is a biologically pure culture of a bacterium. In still another embodiment, the DFM is a composition that comprises at least two bacterial strains that contain no other microorganisms. In still another embodiment, the DFM is a composition that comprises at least two bacterial strains that contain no other microorganisms found in a native environment.

In one embodiment, the DFM may be a viable or inviable microorganism which is used in isolated or semi-isolated form. The DFM may be used in combination with or without the growth medium in which it was cultured.

In one embodiment, the DFM is capable of producing colony forming units when grown on an appropriate media. The appropriate media may comprise (or consist of) a feed or a feed constituent.

In one embodiment, the DFM is incapable of producing colony forming units when grown on an appropriate media. Irrespective of whether the DFM is capable or incapable of producing colony forming units when grown on an appropriate media—the cells may be still metabolically active (e.g. even if they are unable to divide).

In one embodiment the DFM may be administered as inviable cells.

In one embodiment the DFM may be a Bacillus subtilis. In one embodiment the DFM may be selected from the group consisting of: Bacillus subtilis 747, 1104, 1541, 1781, 1999, 2018.

In one embodiment, the DFM is a multi-strain DFM comprising Bacillus subtilis 747, 1104, 1541, 1781, 1999, 2018.

a. Formulation of a DFM

In one embodiment, one or more carrier(s) or other ingredients can be added to the DFM. The DFM may be presented in various physical forms, for example, as a top dress, as a water soluble concentrate for use as a liquid drench or to be added to a milk replacer, gelatin capsule, or gels.

In one embodiment of the top dress form, freeze-dried fermentation product is added to a carrier, such as whey, maltodextrin, sucrose, dextrose, limestone (calcium carbonate), rice hulls, yeast culture, dried starch, and/or sodium silico aluminate.

In one embodiment of the water soluble concentrate for a liquid drench or milk replacer supplement, freeze-dried fermentation product is added to a water soluble carrier, such as whey, maltodextrin, sucrose, dextrose, dried starch, sodium silico aluminate, and a liquid is added to form the drench or the supplement is added to milk or a milk replacer.

In one embodiment of the gelatin capsule form, freeze-dried fermentation product is added to a carrier, such as whey, maltodextrin, sugar, limestone (calcium carbonate), rice hulls, yeast culture dried starch, and/or sodium silico aluminate.

In one embodiment, the bacteria and carrier are enclosed in a degradable gelatin capsule. In one embodiment of the gels form, freeze-dried fermentation product is added to a carrier, such as vegetable oil, sucrose, silicon dioxide, polysorbate 80, propylene glycol, butylated hydroxyanisole, citric acid, ethoxyquin, and/or artificial coloring to form the gel.

The DFM(s) may optionally be admixed with a dry formulation of additives including but not limited to growth substrates, enzymes, sugars, carbohydrates, extracts and growth promoting micro-ingredients. The sugars could include the following: lactose; maltose; dextrose; malto-dextrin; glucose; fructose; mannose; tagatose; sorbose; raffinose; and galactose. The sugars range from 50-95%, either individually or in combination. The extracts could include yeast or dried yeast fermentation solubles ranging from 5-50%. The growth substrates could include: trypticase, ranging from 5-25%; sodium lactate, ranging from 5-30%; and, Tween 80, ranging from 1-5%. The carbohydrates could include mannitol, sorbitol, adonitol and arabitol. The carbohydrates range from 5-50% individually or in combination. The micro-ingredients could include the following: calcium carbonate, ranging from 0.5-5.0%; calcium chloride, ranging from 0.5-5.0%; dipotassium phosphate, ranging from 0.5-5.0%; calcium phosphate, ranging from 0.5-5.0%; manganese proteinate, ranging from 0.25-1.00%; and, manganese, ranging from 0.25-1.0%.

To prepare DFMs described herein, the culture(s) and carrier(s) (where used) can be added to a ribbon or paddle mixer and mixed for about 15 minutes, although the timing can be increased or decreased. The components are blended such that a uniform mixture of the cultures and carriers result. The final product is preferably a dry, flowable powder. The DFM(s) or composition comprising same can then be added to animal feed or a feed premix, added to an animal's water, or administered in other ways known in the art (preferably simultaneously with the enzymes of the present invention). A feed for an animal can be supplemented with one or more DFM(s) described herein or with a composition described herein.

In one embodiment, the DFMs and compositions disclosed herein may be in the form of a concentrate. Typically these concentrates comprise a substantially high concentration of a DFM.

Powders, granules and liquid compositions in the form of concentrates may be diluted with water or resuspended in water or other suitable diluents, for example, an appropriate growth medium such as milk or mineral or vegetable oils, to give compositions ready for use.

The DFM and compositions disclosed herein in the form of concentrates may be prepared according to methods known in the art.

b. Dosing

In one embodiment, DFMs and compositions disclosed herein provide a content of viable cells (colony forming units, CFUs) in the range selected of about 10⁸ CFU/head/day to about 5×10⁹CFU/head/day.

In one embodiment, a DFM in the form of a concentrate may have a content of viable cells in the range of at least 10⁹ CFU/g to about 10¹² CFU/g, or at least 10¹¹ CFU/g to about 10¹² CFU/g.

In one embodiment, the DFM and/or feed additive composition disclosed herein may be designed for one-time dosing or may be designed for feeding on a daily basis. The optimum amount of the composition (and each component therein) to be used in the combination will depend on the product to be treated and/or the method of contacting the product with the composition and/or the intended use for the same. The amount of DFM used in the compositions should be a sufficient amount to be effective and to remain sufficiently effective in improving the performance of the animal fed feed products containing said composition. This length of time for effectiveness should extend up to at least the time of utilization of the product (e.g. feed additive composition or feed containing same).

C. Deposits Under the Budapest Treaty

Bacillus strains 747, 1104, 1541, 1781 and 2018 were deposited on May 24, 2016 at the Agricultural Research Service Culture Collection (NRRL), 1815 North University Street, Peoria, Ill., 61604 and given accession numbers NRRL B-67257 for strain 747, NRRL B-67258 for strain 1104, NRRL B-67260 for strain 1541, NRRL B-67259 for strain 1781 and NRRL B-67261 for strain 2018. Strain 1999 was deposited on Sep. 15, 2016 and given the accession number NRRL B-67318. All deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

D. Methods of Culturing Strains

The Bacillus strains can be produced by fermentation of the bacterial strains. Fermentation can be started by scaling-up a seed culture. This involves repeatedly and aseptically transferring the culture to a larger and larger volume to serve as the inoculum for the fermentation, which is carried out in large stainless steel fermentors in medium containing proteins, carbohydrates, and minerals necessary for optimal growth. A non-limiting exemplary medium is Tyticase Soy Broth (TSB). After the inoculum is added to the fermentation vessel, the temperature and agitation are controlled to allow maximum growth. Once the culture reaches a maximum population density, the culture is harvested by separating the cells from the fermentation medium. This is commonly done by centrifugation.

The count of the culture can then be determined. A colony forming unit (CFU) is the viable cell count of a sample resulting from standard microbiological plating methods. The term is derived from the fact that a single cell when plated on appropriate medium will grow and become a viable colony in the agar medium. Since multiple cells may give rise to one visible colony, the term colony forming unit is a more useful unit measurement than cell number.

In one embodiment, the Bacillus strains disclosed herein can be fermented between 5×10⁸ CFU/ml to about 5×10¹¹ CFU/ml.

In at least one embodiment, a level of 2×10⁹ CFU/ml is used. The bacteria are harvested by centrifugation, and the supernatant is removed. The supernatant can be used in the methods described herein. In at least some embodiments, the bacteria are pelleted. In at least some embodiments, the bacteria are freeze-dried. In at least some embodiments, the bacteria are mixed with a carrier. However, it is not necessary to freeze-dry the Bacillus before using them. The strains can also be used with or without preservatives, and in concentrated, unconcentrated, or diluted form.

In one embodiment, the disclosure relates to a biologically pure culture comprising, consisting of, or consisting essentially of one or more Bacillus strains disclosed herein at a concentration of about 5×10² CFU/ml to about 5×10⁹ CFU/ml.

In one embodiment, the disclosure relates to a culture comprising, consisting of, or consisting essentially of one or more Bacillus strains disclosed herein at a concentration selected from the group consisting of 5×10¹¹ CFU/ml, 5×10¹² CFU/ml, and 5×10¹³ CFU/ml.

In one embodiment, the disclosure relates to a culture comprising, consisting of, or consisting essentially of one or more Bacillus strains disclosed herein at a concentration of 5×10¹⁰CFU/ml to 10¹²CFU/ml or 5×10¹¹CFU/ml to 10¹²CFU/ml.

II. Compositions

In one embodiment, the disclosure relates to a composition comprising one or more Bacillus strains disclosed herein. In yet another embodiment, the disclosure relates to a composition comprising one or more SCMs. In yet another embodiment, the disclosure relates to a composition comprising one or more DFMs.

In one embodiment, the disclosure relates to a composition comprising one or more Bacillus strain selected from the group consisting of Bacillus subtilis 781 and Bacillus subtilis 747, wherein the composition is free of other microbial organisms.

In one embodiment, the disclosure relates to a composition comprising one or more Bacillus strain selected from the group consisting of Bacillus subtilis 747, 1781, 1104, 1541, 1999 and 2018, wherein the Bacillus strains are biologically pure prior to formation of the composition.

In one embodiment, the disclosure relates to a composition comprising one or more Bacillus strain selected from the group consisting of Bacillus subtilis 1781, and Bacillus subtilis 747; (b) a carrier and (c) a preservative. In another embodiment, one or more of the Bacillus strains are at a concentration of at least 10⁹ CFU/ml.

In another embodiment, the disclosure relates to a composition comprising (a) one or more Bacillus strain selected from the group consisting of Bacillus subtilis 1781, Bacillus subtilis 747, and (b) a feed. In yet another embodiment, the disclosure relates to a feed comprising (a) one or more Bacillus strain selected from the group consisting of Bacillus subtilis 1104, 1781 and 2018 (b) silage or fodder, wherein the feed has a lower concentration of Clostridia as compared to a feed lacking the Bacillus strains.

III. Feed/Feedstuff

In one embodiment, the strains, SCMs, DFMs, and compositions disclosed herein may be used as—or in the preparation of—a feed. In one embodiment, the feed is fodder. In another embodiment, the feed is silage.

Forage that has been grown while still green and nutritious can be conserved through a natural ‘pickling’ process. Lactic acid is produced when the sugars in the forage plants are fermented by bacteria in a sealed container (‘silo’) with no air. Forage conserved this way is known as ‘ensiled forage’ or ‘silage’ and will keep for up to three years without deteriorating. Silage is very palatable to livestock and can be fed at any time.

The feed may be in the form of a solution or as a solid—depending on the use and/or the mode of application and/or the mode of administration. When used as—or in the preparation of—a feed—such as functional feed—the composition of the present invention may be used in conjunction with one or more of: a nutritionally acceptable carrier, a nutritionally acceptable diluent, a nutritionally acceptable excipient, a nutritionally acceptable adjuvant, a nutritionally active ingredient.

In one embodiment, the strains, SCMs, DFMs, and compositions disclosed herein are admixed with a feed component to form a feedstuff. In one embodiment, the feed may be a fodder, or a premix thereof, a compound feed, or a premix thereof. In one embodiment, the strains, SCMs, DFMs, and compositions disclosed herein may be admixed with a compound feed, a compound feed component or to a premix of a compound feed or to a fodder, a fodder component, or a premix of a fodder.

In still another embodiment, the strains, SCMs, DFMs, and compositions disclosed herein can me mixed with silage, compressed and pelleted feeds, oils and mixed rations, and also sprouted grains and legumes. Fodder may be obtained from one or more of the plants selected from: barley rapeseed (canola), corn (maize), millet, oats, sorghum, soybeans, wheat, and legumes.

Any feedstuff disclosed herein may comprise one or more feed materials selected from the group comprising a) cereals, such as small grains (e.g., wheat, barley, rye, oats and combinations thereof) and/or large grains such as maize or sorghum; b) by products from cereals, such as corn gluten meal, wet-cake (particularly corn based wet-cake), Distillers Dried Grain (DDG) (particularly corn based Distillers Dried Grain (cDDG)), Distillers Dried Grain Solubles (DDGS) (particularly corn based Distillers Dried Grain Solubles (cDDGS)), wheat bran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp; c) protein obtained from sources such as soya, sunflower, peanut, lupin, peas, fava beans, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; d) oils and fats obtained from vegetable and animal sources; e) minerals and vitamins.

A feedstuff may contain at least 10%, at least 20%, at least 30% or at least 50% by weight corn and soybean meal or corn and full fat soy, or wheat meal or sunflower meal.

A feedstuff may contain between about 0 to about 40% corn DDGS. If the feedstuff contain any corn DDGS it may contain between about 5 to about 40% corn DDGS. For poultry—where corn DDGS is present the feedstuff on average may contain between about 7 to 15% corn DDGS. For swine (pigs)—where corn DDGS is present the feedstuff may contain on average 5 to 40% corn DDGS.

A feedstuff may contain corn as a single grain, in which case the feedstuff may comprise between about 35% to about 80% corn.

In one embodiment, the feed may be one or more of the following: a compound feed and premix, including pellets, nuts or (cattle) cake; a crop or crop residue: corn, soybeans, sorghum, oats, barley, copra, chaff, sugar beet waste; fish meal; meat and bone meal; molasses; oil cake and press cake; oligosaccharides; conserved forage plants: silage; seaweed; seeds and grains, either whole or prepared by crushing, milling etc.; sprouted grains and legumes; yeast extract.

In one embodiment, a bacterial strain, SCM, DFM, or composition disclosed herein is admixed with the product (e.g. feedstuff). In another embodiment, a bacterial strain, a SCM, DFM, or a composition may be included in the emulsion or raw ingredients of a feedstuff. For some applications, it is important that the composition is made available on or to the surface of a product to be affected/treated. This allows the composition to impart one or more of the following favourable characteristics: performance benefits.

In one embodiment, a bacterial strain, a SCM, DFM, or a composition disclosed herein may be applied to intersperse, coat and/or impregnate a product (e.g. feedstuff or raw ingredients of a feedstuff).

In one embodiment, a bacterial strain, a SCM, a DFM, or a composition disclosed herein can be added in suitable concentrations—such as for example in concentrations in the final feed product which offer a daily dose of from about 2×10⁵ CFU to about 2×10¹¹ CFU, suitably from about 2×10⁶ to about 1×10¹⁰, or between about 3.75×10⁷ CFU to about 1×10¹⁰ CFU.

In yet another embodiment, a bacterial strain, a SCM, a DFM, or a composition will be thermally stable to heat treatment up to about 70° C.; up to about 85° C.; or up to about 95° C. The heat treatment may be performed for up to about 1 minute; up to about 5 minutes; up to about 10 minutes; up to about 30 minutes; up to about 60 minutes. The term thermally stable means that at least about 75% of the bacterial strain or SCM that were present/active in the additive before heating to the specified temperature are still present/active after it cools to room temperature. In one embodiment, at least about 80% of the bacterial strain or SCM that were present and active in the additive before heating to the specified temperature are still present and active after it cools to room temperature.

In one embodiment, a bacterial strain, a SCM, a DFM, or a composition disclosed herein are homogenized to produce a powder.

IV. Forms

In one embodiment, a bacterial strain, a SCM, a DFM, or a composition and other components and/or the feedstuff comprising same may be used in any suitable form. A bacterial strain, a SCM, a DFM, or a composition disclosed herein may be used in the form of solid or liquid preparations or alternatives thereof. Examples of solid preparations include powders, pastes, boluses, capsules, pellets, tablets, dusts, and granules which may be wettable, spray-dried or freeze-dried. Examples of liquid preparations include, but are not limited to, aqueous, organic or aqueous-organic solutions, suspensions and emulsions.

In some applications, a bacterial strain, a SCM, a DFM, or a composition disclosed herein may be mixed with feed or administered in the drinking water. In one embodiment the dosage range for inclusion into water is about 1×10⁸ CFU/animal/day to about 1×10¹⁰ CFU/animal/day, and more preferably about 1×10⁹ CFU/animal/day.

Suitable examples of forms include one or more of: powders, pastes, boluses, pellets, tablets, pills, capsules, ovules, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.

By way of example, if a bacterial strain, a SCM, a DFM, or a composition disclosed herein is used in a solid, e.g. pelleted form, it may also contain one or more of: excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine; disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates; granulation binders such as polyvinylpyrrolidone, hydroxypropylmethyl cellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia; lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Examples of nutritionally acceptable carriers for use in preparing the forms include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.

In one embodiment, excipients for the forms include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols.

For aqueous suspensions and/or elixirs, a bacterial strain, a SCM, a DFM, or a composition disclosed herein may be combined with various sweetening or flavoring agents, coloring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, propylene glycol and glycerin, and combinations thereof.

In one embodiment, non-hydroscopic whey can be used as a carrier for a bacterial strain, a SCM, a DFM, or a composition disclosed herein (particularly bacterial DFMs) and is a good medium to initiate growth. A bacterial strain, a SCM, a DFM, or a composition disclosed herein containing pastes may be formulated with vegetable oil and inert gelling ingredients.

In one embodiment, fungal products may be formulated with grain by-products as carriers.

The dry powder or granules may be prepared by means known to those skilled in the art, such as, in top-spray fluid bed coater, in a bottom spray Wurster or by drum granulation (e.g. High sheer granulation), extrusion, pan coating or in a microingredients mixer.

In another embodiment, the bacterial strains, SCMs, DFMs or compositions disclosed herein may be coated, for example encapsulated. In some embodiments, such as where the bacterial strain is capable of producing endospores, the bacterial strains, SCMs, DFMs or compositions disclosed herein may be provided without any coating.

V. Methods of Treating Feed with an DFM

In one embodiment, the disclosure relates to methods of reducing spoilage of feed comprising mixing a SCM or composition disclosed herein with feed in an effective amount to reduce spoilage of silage in comparison to feed not mixed with the SCM or composition. In one embodiment, the feed is silage or fodder.

In one embodiment, spoilage of feed is reduced by a percentage selected from the group consisting of: at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least, at least 95%, and at least 99% as compared to feed not treated with an SCM or composition.

In one embodiment, spoilage of the feed is reduced from 2 to 5%, or from 5 to 10%, or from 10 to 15%, or from 15 to 20%, or from 20 to 25%, or from 25 to 30%, or from 30 to 35%, or from 35 to 40%, or from 40 to 45%, or from 45 to 50%, or from 50 to 55%, or from 55 to 60%, or from 60 to 65%, or from 65 to 70%, or from 70 to 75%, or from 75 to 80%, or from 80 to 85%, or from 85 to 90%, or from 90 to 95%, or from 95 to 99% as compared to feed not treated with an SCM or composition.

In another embodiment, the disclosure relates to methods of controlling growth of microorganisms in feed comprising mixing an SCM or composition disclosed herein with feed in an effective amount to control growth of microorganisms in feed.

In another embodiment, the disclosure relates to methods of reducing growth of clostridia in feed comprising mixing an SCM or composition disclosed herein with feed in an effective amount to control growth of clostridia in feed.

In another embodiment, the disclosure relates to methods of reducing growth of clostridia in fodder comprising: (a) mixing an SCM or composition disclosed herein in a liquid; (b) mixing the liquid with fodder; and (c) placing the fodder in a sealed container.

In another embodiment, the disclosure relates to methods of reducing growth of clostridia in fodder comprising: (a) mixing an SCM or composition disclosed herein in a dry form with fodder; and (b) placing the fodder in a sealed container.

In another embodiment, the disclosure relates to methods of reducing growth of clostridia in fodder comprising: (a) spraying an SCM or composition disclosed herein onto fodder; and (b) placing the fodder in a sealed container.

In one embodiment, the disclosure relates to methods of producing silage comprising: (a) mixing an SCM or composition disclosed herein in a liquid; (b) mixing the liquid with fodder; (c) placing the fodder in a sealed container for a suitable period of time; and (d) obtaining silage from the container.

In another embodiment, the disclosure relates to methods of producing silage comprising: (a) mixing an SCM or composition disclosed herein in a dry form with fodder; (b) placing the fodder in a sealed container; and (c) obtaining or harvesting silage from the container.

In another embodiment, the disclosure relates to methods of producing silage comprising: (a) spraying an SCM or composition disclosed herein onto fodder; (b) placing the fodder in a sealed container; and (c) obtaining or harvesting silage from the container.

In one embodiment, the SCM or composition is mixed or sprayed with a percentage of the fodder selected from the group consisting of: 5-10%, 10-220%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, and 95-99% and 100% of the fodder.

In one embodiment, the SCM or composition is mixed or sprayed with at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% and 100%.

In one embodiment, the sealed container is a silo. In another embodiment, the sealed container is a plastic bag.

In one embodiment, the silage has a lower concentration of pathogenic microorganisms, such as clostridia, as compared to silage obtained from fodder not treated with Bacillus strains.

In one embodiment, growth of clostridia is reduced by a percentage selected from the group consisting of: at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least, at least 95%, and at least 99% as compared to feed not treated with an SCM or composition.

In one embodiment, growth of clostridia is reduced from 2 to 5%, or from 5 to 10%, or from 10 to 15%, or from 15 to 20%, or from 20 to 25%, or from 25 to 30%, or from 30 to 35%, or from 35 to 40%, or from 40 to 45%, or from 45 to 50%, or from 50 to 55%, or from 55 to 60%, or from 60 to 65%, or from 65 to 70%, or from 70 to 75%, or from 75 to 80%, or from 80 to 85%, or from 85 to 90%, or from 90 to 95%, or from 95 to 99% as compared to feed not treated with an SCM or composition.

In one embodiment, the clostridia is Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Clostridium acetobutylicum, Clostridium difficile, Clostridium novyi

In one embodiment, the clostridia is Clostridium perfringens.

In another embodiment, the disclosure relates to methods of increasing the shelf life or storage of feed comprising mixing an SCM or composition with feed in an effective amount to increase the shelf life or storage duration of feed in comparison to feed not mixed with an SCM or composition.

In one embodiment, the SCM comprises one or more Bacillus strains. In one embodiment, the Bacillus strains are at a concentration selected from the group consisting of: 5,000 CFU/g, 10,000 CFU/g, 15,000 CFU/g, 20,000 CFU/g, 25,000 CFU/g, 30,000 CFU/g, 35,000 CFU/g, 40,000 CFU/g, 45,000 CFU/g, 50,000 CFU/g, 55,000 CFU/g, 60,000 CFU/g, 65,000 CFU/g, 70,000 CFU/g, 75,000 CFU/g, 80,000 CFU/g, 85,000 CFU/g, 90,000 CFU/g, 95,000 CFU/g, and 100,000 CFU/g. In one embodiment, the SCM is a composition of Bacillus amyloliquefaciens 1104 and Bacillus subtilis 1781.

In yet another embodiment, the Bacillus strains are at a concentration selected from the group consisting of: from 5,000 CFU/g to 10,000 CFU/g.

In yet another embodiment, the Bacillus strains are at a concentration selected from the group consisting of: from 10,000 CFU/g to 75,000 CFU/g.

In yet another embodiment, the Bacillus strains are at a concentration selected from the group consisting of: 10³CFU/g, 10⁴ CFU/g, 10⁵ CFU/g, 10⁶ CFU/g, 10⁷ CFU/g, 10⁸ CFU/g, 10⁹ CFU/g, and 10¹⁰ CFU/g.

In another embodiment, the composition further comprises a preservative.

In one embodiment, the SCM is a composition of lactic acid bacteria and Bacillus. In one embodiment, the CFU of the lactic acid bacteria is 2× the CFUs of the Bacillus strains in the composition. In yet another embodiment, the CFU of the lactic acid bacteria is 3× the CFUs of the Bacillus strains in the composition.

In one embodiment, the concentration of the lactic acid bacteria is selected from the group consisting of 50,000 CFU/g, 75,000 CFU/g, 100,000 CFU/g, 125,000 CFU/g, 150,000 CFU/g, 200,000 CFU/g, 250,000 CFU/g, 275,000 CFU/g, 300,000 CFU/g, 325,000 CFU/g, 350,000 CFU/g, 375,000 CFU/g, and 400,000 CFU/g.

In still another embodiment, the concentration of the Bacillus strain is selected from the group consisting of: 25,000 CFU/g, 50,000 CFU/g, 75,000 CFU/g, 100,000 CFU/g, 125,000 CFU/g, 150,000 CFU/g, and 200,000 CFU/g.

In yet another embodiment, the SCM is a composition comprising Bacillus amyloliquefaciens 1104 and Bacillus subtilis 1781 and one or more of the following strains: Enterococcus faecium, Lactobacillus plantarum LP 115, Pediococcus acidilactici PJ300 and Pediococcus pentosaceus P751. In one embodiment, the concentration of the Bacillus strains in the composition is from about 25,000 CFU/g to about 75,000 CFU/g. In another embodiment, the concentration of the lactic acid bacteria is from about 75,000 CFU/g to about 225,000 CFU/g.

VI. Methods of Administering DFMs to an Animal

In one embodiment, the disclosure relates to methods of increasing performance metrics of an animal. In another embodiment, the disclosure relates to methods of increasing performance metrics of a ruminant.

In yet another embodiment, the disclosure relates to a method comprising administering to an animal a composition comprising a DFM. In still another embodiment, the disclosure relates to a method comprising administering to an animal an effective amount of a composition comprising DFMs to increase performance of the animal. This effective amount can be administered to the animal in one or more doses.

In another embodiment, the disclosure relates to a method comprising administering to an animal an effective amount of a composition comprising DFMs to increase average daily feed intake.

In another embodiment, the disclosure relates to a method comprising administering to an animal an effective amount of a composition comprising DFMs to increase average daily weight gain.

In another embodiment, the disclosure relates to a method comprising administering to an animal an effective amount of a composition comprising DFMs to increase total weight gain.

In another embodiment, the disclosure relates to a method comprising administering to an animal an effective amount of a composition comprising DFMs to increase feed conversion, which can be measured by either feed:gain or gain:feed.

In another embodiment, the disclosure relates to a method comprising administering to an animal an effective amount of a composition comprising DFMs to increase feed efficiency.

In another embodiment, the disclosure relates to a method comprising administering to an animal an effective amount of a composition comprising DFMs and exogenous feed enzymes to decrease mortality.

In another embodiment, the disclosure relates to a method comprising administering to an animal an effective amount of a composition comprising DFMs to decrease actual production costs.

In still another embodiment, the DFM is Bacillus subtilis 1104 or a strain having all of the identifying characteristics of Bacillus subtilis 1104. In still another embodiment, the DFM is Bacillus subtilis 1781 or a strain having all of the identifying characteristics of Bacillus subtilis 1781. In still another embodiment, the DFM is Bacillus subtilis 747 or a strain having all of the identifying characteristics of Bacillus subtilis 747. In still another embodiment, the DFM is Bacillus subtilis 1541 or a strain having all of the identifying characteristics of Bacillus subtilis 1541. In still another embodiment, the DFM is Bacillus subtilis 1999 or a strain having all of the identifying characteristics of Bacillus subtilis 1999. In still another embodiment, the DFM is Bacillus subtilis 2018 or a strain having all of the identifying characteristics of Bacillus subtilis 2018.

In still another embodiment, the DFM is a multi-strain comprising Bacillus subtilis 747 and Bacillus subtilis 1781.

In some embodiments, the one or more Bacillus strain(s) is (are) added to an animal's feed at a rate of at least 1×10⁹ CFU/animal/day. In some embodiments, the one or more Bacillus strain(s) is(are) fed at about 1×10⁹ CFU/g feed to about 1×10¹⁰ CFU/g feed.

The DFM provided herein can be administered, for example, as the strain-containing culture solution, the strain-containing supernatant, or the bacterial product of a culture solution.

Administration of a DFM or a composition disclosed herein to an animal can increase the performance of the animal. In one embodiment, administration of a DFM provided herein to an animal can increase the average daily feed intake (ADFI), average daily gain (ADG), or feed efficiency (gain:feed; G:F) (collectively, “performance metrics”). One or more than one of these performance metrics may be improved.

The composition comprising DFMs may be administered to the animal in one of many ways. For example, the composition can be administered in a solid form as a veterinary pharmaceutical, may be distributed in an excipient, preferably water, and directly fed to the animal, may be physically mixed with feed material in a dry form, or the composition may be formed into a solution and thereafter sprayed onto feed material. The method of administration of the compositions disclosed herein to the animal is considered to be within the skill of the artisan.

When used in combination with a feed material, the feed material for ruminants can be grain or hay or silage or grass, or combinations thereof. Included amongst such feed materials are corn, dried grain, alfalfa, any feed ingredients and food or feed industry by-products as well as bio fuel industry by-products and corn meal and mixtures thereof. For monogastric diets, the feed material can include corn, soybean meal, byproducts like distillers dried grains with solubles (DDGS), and vitamin/mineral supplement. Other feed materials can also be used. Administration is possible at any time with or without feed. However, the bacterium is preferably administered with or immediately before feed.

Thus, in at least some embodiments, the effective amount of the composition comprising DFMs is administered to an animal by supplementing a feed intended for the animal. As used herein, “supplementing,” refers to the action of incorporating the effective amount of bacteria provided herein directly into the feed intended for the animal. Thus, the animal, when feeding, ingests the bacteria provided herein. In one embodiment, the disclosure relates to Bacillus strains. In one embodiment the Bacillus strain is Bacillus subtilis 1104. In still another embodiment, the Bacillus strain is Bacillus subtilis 1781. In still another embodiment, the Bacillus strain is Bacillus subtilis 747. In still another embodiment, the Bacillus strain is Bacillus subtilis 1541. In still another embodiment, the Bacillus strain is Bacillus subtilis 2018.

In one embodiment, the disclosure relates to a composition comprising two or more of the following Bacillus strains: Bacillus subtilis 1104, Bacillus subtilis 1781, Bacillus subtilis 747, Bacillus subtilis 1541, Bacillus subtilis 1999, and Bacillus subtilis 2018.

In still another embodiment, the disclosure relates to a composition comprising Bacillus subtilis 747 and one or more of the following Bacillus strains: Bacillus subtilis 1104, Bacillus subtilis 1781, Bacillus subtilis 1541, Bacillus subtilis 1999, and Bacillus subtilis 2018.

In still another embodiment, the disclosure relates to a composition comprising Bacillus subtilis 1781 and one or more of the following Bacillus strains: Bacillus subtilis 1104, Bacillus subtilis 747, Bacillus subtilis 1541, Bacillus subtilis 1999, and Bacillus subtilis 2018.

In yet another embodiment, the disclosure relates to a composition comprising Bacillus subtilis 747 and Bacillus subtilis 1781.

In yet another embodiment, the disclosure relates to a composition comprising one or more Bacillus strains selected from the group consisting of: Bacillus subtilis 1104, Bacillus subtilis 1781, and Bacillus subtilis 747, Bacillus subtilis 1541, Bacillus subtilis 1999, Bacillus subtilis 2018 and a preservative.

In yet another embodiment, the disclosure relates to a composition comprising one or more Bacillus strains selected from the group consisting of: Bacillus subtilis 1104, Bacillus subtilis 1781, and Bacillus subtilis 747, Bacillus subtilis 1541, Bacillus subtilis 1999, Bacillus subtilis 2018 and Lactobacillus plantarum.

In another embodiment, the disclosure relates to Bacillus strains, compositions, and methods for controlling or reducing spoilage of a feed, including but not limited to fodder and silage.

In another embodiment, the disclosure relates to Bacillus strains, compositions and methods for controlling or reducing growth of microorganisms. In another embodiment, compositions and methods are disclosed for controlling or reducing growth of microorganisms in a feed, including but not limited to silage and/or fodder.

In still another embodiment, Bacillus strains, compositions and methods are disclosed for controlling or reducing growth of Clostridia. In still another embodiment, Bacillus strains, compositions and methods are disclosed for controlling or reducing growth of Clostridia in feed including but not limited to silage and/or fodder.

In one embodiment, the disclosure relates to a composition comprising one or more spoilage control microbials. In another embodiment, the disclosure relates to a composition comprising one or more spoilage control microbials and one or more additional component(s).

In another embodiment, the disclosure relates to Bacillus strains, compositions and methods for improving the performance of an animal. In another embodiment, the disclosure relates to one or more direct fed microbials for improving the performance of an animal.

In another embodiment, the disclosure relates to a method for increasing the shelf life of silage or fodder comprising: mixing an effective amount of at least one Bacillus strain with silage to increase the shelf life of the silage or fodder. In still another embodiment, the disclosure relates to a method for increasing the shelf life of silage or fodder comprising: mixing a composition comprising at least one spoilage control microbial with silage to increase the shelf life of the silage or fodder. In still another embodiment, the disclosure relates to a method for increasing the shelf life of silage or fodder comprising: mixing a composition comprising at least two spoilage control microbials with silage to increase the shelf life of the silage or fodder.

In yet another embodiment, the disclosure relates to a method of controlling growth of a microorganism in silage or fodder comprising mixing an effective amount of at least one Bacillus strain with the silage or fodder. In one embodiment, the Bacillus strain is mixed with the silage at the time of ensiling.

In still another embodiment, the disclosure relates to a method of controlling growth of a microorganism in silage or fodder comprising: mixing a composition comprising at least one spoilage control microbial with silage or fodder to control growth of a microorganism in the silage or fodder. In still another embodiment, the disclosure relates to a method of controlling growth of a microorganism in silage or fodder comprising: mixing a composition comprising at least two spoilage control microbials with silage or fodder.

In yet another embodiment, the disclosure relates to a method for improving performance of an animal comprising administering one or more Bacillus strains, one or more direct fed microbials or a composition to an animal to improve performance of said animal in comparison to an animal not fed the Bacillus strain, the direct fed microbial or the composition.

In one embodiment, the disclosure relates to a method of controlling, or treating or preventing growth of a pathogen in an animal comprising administering one or more Bacillus strains, one or more direct fed microbials or a composition to an animal to control, treat, or prevent growth of a pathogen as compared to an animal not fed the Bacillus strain, the direct fed microbial or the composition. In one embodiment, the pathogen is Clostridium perfringens.

In still another embodiment, the disclosure relates to a feed for an animal comprising one or more Bacillus strain, one or more DFM or one or more composition.

DESCRIPTION OF A PREFERRED EMBODIMENT Examples Example 1: Selection of Bacillus Strains to Inhibit Clostridium perfringens and Non-Toxigenic Clostridia Isolated from Feed Samples

Introduction:

The ensiling process is a means of preserving the nutritional value of a moist crop by promoting anaerobic fermentation of the sugars present in the crop and converting them to lactic acid and other beneficial acidic compounds that preserve the material (Muck, 2010). The moist crop can support the growth of a variety of spoilage microorganisms, such as clostridia, bacilli, yeasts and molds that contribute to the degradation of nutrient value particularly when anaerobic conditions are not maintained or when the lactic acid end-product of anaerobic fermentation is inadequate to sufficiently decrease pH (Driehuis and Oude Elferink, 2000). Therefore, the ensiling process allows for long-term storage of feeding material for ruminant livestock when fresh forage is unavailable.

Because of the many variables that prevent ideal conditions for preserving silage, lactic acid bacteria are often utilized as silage inoculants to promote proper fermentation and optimal preservation of silage. Lactic acid bacteria grow quickly in anaerobic conditions and become the dominant microorganisms present in the crop, and lower the pH through the production of lactic acid, their fermentation end product. Enterobacteria and bacilli are controlled by lowering the pH to less than 5, whereas clostridia are more difficult to inhibit as some can grow at a lower pH (Driehuis, 2013). Therefore a lower pH may be needed to preserve the crop and prevent the growth of clostridial spoilage organisms when conditions are less optimal for lactic acid bacteria fermentation, such as in conditions of high moisture (Driehuis and Oude Elferink, 2000). These spoilage clostridia tend to have their negative effects on silage quality after the lactic acid bacteria have ceased growing.

Controlling clostridia organisms in silage is important to prevent the detrimental effects these bacteria have on silage quality. Generally, clostridia spoilage organisms are categorized into three groups including proteolytic clostridia that ferment amino acids and produce ammonia, amines, and carbon dioxide, the Clostridium butyricum group that ferments carbohydrates, and the C. tyrobutyricum group that ferments sugars and lactic acid, the latter two groups producing butyric acid, acetic acid, hydrogen, and carbon dioxide as end products (Muck, 2010). Clostridia activity in silage is undesirable due to the reduced intake observed in cattle when clostridial activity is present and because of the reduced nutritional quality of the silage that results from clostridial fermentation. The fermentation of lactic acid to butyric acid by the butyrate producing clostridia results in approximately 50% loss in dry matter and 18% loss in gross energy from the silage feedstuff (McDonald et al., 1991). Furthermore, clostridia spoilage organisms have a detrimental effect on the health of the cattle as evidenced by greater incidence of acidosis when cattle are fed clostridial silage (Seglar, 2003).

Clostridia fermentation in silage is controlled using lactic acid bacteria as silage inoculants to support the preservation of the crop and by ensuring the crop is harvested and ensiled under low-moisture conditions. However, managing the on-farm conditions such as weather that would impact the moisture content of the crop at harvest is not always practical or possible, and often ensiling occurs under sub-optimal conditions. Although bacilli are considered silage spoilage organisms, members of the Bacillus genera are known to produce antimicrobial compounds capable of inhibiting competing bacteria in the surrounding environment, and have demonstrated efficacy in controlling the growth of clostridia Bacilli usually result in accelerating the spoilage of silage following exposure to oxygen, but rarely impact fermentation of the crop under the anaerobic conditions of the silo (Muck, 2010). Therefore, Bacillus strains are needed that can be added at the time of ensiling to control the growth of clostridia spoilage organisms in silage harvested under high-moisture conditions.

Materials and Methods:

Forage samples were gathered from 111 different dairies. Samples were diluted 1:10 with sterile peptone, heat shocked for 10 minutes at 50° C., enumerated in sterile peptone and pour plated on Tryptose Sulphite Cycloserine (TSC) agar with D-cycloserine (400 mg/L) to select for clostridia. Agar plates were incubated at 37° C. anaerobically for 24 hours for clostridia growth. If present, isolated sulphite-reducing colonies were picked into Reinforced Clostridia Medium (RCM) (Oxoid, CM0149) and incubated anaerobically for 24 hours at 37° C. After 24 hours of incubation the cultures were transferred (10%) to Brain Heart Infusion (BHI) broth (BD, 211059) and incubated anaerobically for 24 hours at 37° C.

DNA extractions of the cultures were performed in 96-well blocks containing 500 μl presumptive clostridia culture per well. Cells were harvested by centrifugation at 4,700 rpm for 10 minute and the supernatant was discarded. Cells were re-suspended in 500 μl of 50 mM of EDTA-2Na (pH=8.0). Aliquots of 300 μl of the suspended cells were transferred to a new 96-well block and combined with 20 μl of lysozyme from chicken egg white (Sigma, L6876) solution (100 mg/ml in 50 mM EDTA) to lyse bacterial cells. The 96-well block was incubated for 1 hour at 37° C. to lyse bacterial cells. Following the incubation 220 μl of lysis buffer (6 M Guanidine, 20% Triton-X 100, 10 mM Tris-HCL, pH 7.5) was added, mixed then incubated at room temperature for 15 minutes. Following the incubation 20 μl of Proteinase K (NEB, 800 U/mL) was added to each well, mixed and incubated at 55° C. for 30 minutes to degrade proteins. The cell lysate was then transferred to 96-well binding plate (Promega, A2278) and centrifuged at 4,700 rpms for 5 minutes. Flow through was discarded, three washes of the binding plate columns were executed by centrifuging 750 μl of Column Wash Solution (Promega, A1318) at 4700 rpms for 1 minute and 30 seconds and discarding the flow through at the end of each spin. The binding plate was centrifuged for an additional 10 minutes at 4,700 rpm to remove any residual ethanol. A clean elution plate was then placed under the binding plate and DNA was eluted with 200 μl of Nuclease Free Water (Promega, P1195) pre-warmed to 55° C.

DNA was screened for toxin genes (α, β, ε, and ι) specific to C. perfringens using polymerase chain reaction (PCR). Amplification of toxin genes was executed using a multiplex PCR containing four primer sets (Yoo et al., 1997) (Table 1). The PCR mixture contained 2.5 μl 10×PCR Buffer, 2 μl of 50 mM MgCl₂, 0.5 μM of each primer, 0.1 μl of Invitrogen™ Platinum™ Taq DNA Polymerase, 2.5 μl of DNA, and sterile water to achieve 25 μl for a total reaction volume. The mixture underwent 5 minutes at 94° C., followed by 30 cycles of 94° C. for 1 minute, 55° C. for 1 minute, 72° C. for 1 minute finishing with a final elongation of 3 minutes at 72° C. PCR products were observed using a Fragment Analyzer (Advanced Analytics) to determine if amplification was achieved.

TABLE 1 Primers used to amplify C. perfringens toxin genes from the clostridia isolates collected, generate RAPD profiles from the C. perfringens positive isolates, and amplify the 16S rRNA gene from representatives of the non-toxigenic clostridia for DNA sequencing. Primer Nucleotide sequence Alpha Toxin Forward 5′-GTTGATAGCGCAGGACATGTTAAG-3′ (SEQ ID No. 7) Alpha Toxin Reverse 5′-CATGTAGTCATCTGTTCCAGCATC-3′ (SEQ ID No. 8) Beta Toxin Forward 5′-ACTATACAGACAGATCATTCAACC-3′ (SEQ ID No. 9) Beta Toxin Reverse 5′-TTAGGAGCAGTTAGAACTACAGAC-3′ (SEQ ID No. 10) Epsilon Toxin 5′-ACTGCAACTACTACTCATACTGTG-3′ Forward (SEQ ID No. 11) Epsilon Toxin 5′-CTGGTGCCTTAATAGAAAGACTCC-3′ Reverse (SEQ ID No. 12) Iota Toxin Forward 5′-GCGATGAAAAGCCTACACCACTAC-3′ (SEQ ID No. 13) Iota Toxin Reverse 5′-GGTATATCCTCCACGCATATAGTC-3′ (SEQ ID No. 14) RAPD primer 2 GTTTCGCTCC (SEQ ID No. 15) 16S_27F-YM AGAGTTTGATYMTGGCTCAG (SEQ ID No. 16) 16S_1492R-Y TACCTTGTTAYGACTT (SEQ ID No. 17)

Unique strain-specific genetic fingerprints were generated using Random Amplification of Polymorphic DNA (RAPD) analysis to determine diversity among silage clostridia isolates. The PCR contained 5 μl of DNA, 2.5 μl RAPD primer 2 (10 M; Table 4), and 17.5 μl of sterile water which was added to a Ready-To-Go RAPD Analysis Bead (Life Sciences, 27-9500-01). The mixture underwent 5 minutes at 95° C., followed by 45 cycles of 95° C. for 1 minute, 36° C. for 1 minute, 72° C. for 2 minutes, and finishing with a final elongation of 5 minutes at 72° C. The PCR products were observed on a Fragment Analyzer (Advanced Analytics) to determine amplification patterns and were imported into BioNumerics, bioinformatics software for analysis. RAPD patterns were compared with a band based Dice correlation analysis method to determine the similarity between RAPD patterns.

To identify non-toxigenic Clostridium isolates, a PCR reaction was performed on forage samples to amplify the 16S region of rDNA using primers 27F-YM and 1492R-Y (Table 1). The PCR mixture contained 5 μl of 10×PCR Buffer, 2 μl of 50 mM MgCl₂, 1 μl of 50 mM dNTPs, 0.4 μM of each primer (Table 3.), 0.2 μl of Invitrogen™ Platinum™ Taq DNA Polymerase, 5 μl of DNA, and sterile water was added to achieve 50 μl for a total reaction volume. The mixture underwent 4 minutes at 95° C., followed by 35 cycles of 95° C. for 30 seconds, 50° C. for 30 seconds, 72° C. for 2 minutes finishing with a final elongation of 7 minutes at 72° C. A quality check was done on the amplification and PCR product was sent to MWG operon (http://www.operon.com/) to obtain the sequences for the 16S genes. Sequences were compared to known typed bacterial strains obtained from EZbiocloud online electronic database (http://www.ezbiocloud.net/). Based on comparisons of these sequences a bacterial identification was assigned to the isolates.

Antimicrobial screening was done on C. perfringens isolates and non-toxigenic clostridia isolates obtained from feed samples to gauge the effectiveness of the antimicrobial bacteriocin produced by the inventors' identified Bacillus strains 747, 1104, 1541, 1781, and 2018. Strain 1999 was tested against non-toxigenic clostridia isolates. Bacteriocin was harvested by growing each strain at 32° C. in a shaking incubator at 150 rpms for 24 hours in Brain Heart Infusion (BHI) broth. A 1% transfer of the 24 hour culture to fresh BHI broth was executed after incubation. The Bacillus were then incubated for 36-48 hours in a 32° C. shaking incubator at 150 rpms. The culture was then centrifuged at 14,000×g for 20 minutes, supernatant was then filtered with a 0.2 m filter to remove any residual cells.

A bacteriocin turbidity assay was executed by growing clostridia strains isolated from silage samples in Reinforced Clostridial Medium (RCM) for 24 hours, anaerobically, at 37° C. Overnight culture was transferred (1%) to sterile RCM and immediately used in the assay. Four replicates of each clostridia isolate were plated in a sterile 48 well reaction plate. Each clostridia isolate was tested as follows: 600 μl inoculated clostridia culture (positive control), 600 μl RCM+70 μl bacteriocin from Bacillus 2018, 600 μl RCM+70 μl bacteriocin from Bacillus 1104, 600 μl RCM+70 μl bacteriocin from Bacillus 1541, 600 μl RCM+70 μl bacteriocin from Bacillus 1781, 600 μl RCM+70 μl bacteriocin from Bacillus 747, and 670 RCM (negative control). Plates were incubated anaerobically at 37° C. for 24 hours then read using a BioTek Epoch Microplate Spectrophotometer with readings obtained at a wavelength of 600 nm. Before readings, 70 μl of sterile water was added to the positive control to ensure equal volumes in each well. Optical density readings from the negative controls were subtracted from all OD readings and percent inhibition was calculated using the each bacteriocin treatment relative to the positive control.

Results: Clostridia enumeration results, from 1,169 tested feed samples from 111 different locations, indicated the average level of clostridia CFU/g across all samples was 4.2 E+03 CFU/g. Individual samples ranged from <10 to 4.1 E+06 CFU/g (Table 2).

A total of 3,958 presumptive clostridia isolates have been tested for the indicated C. perfringens toxin genes. Isolates tested were harvested from silage samples collected from >100 different agricultural locations. Of the 3,958 isolates screened, 756 isolates (19.1%) tested positive for at least 1 of the toxin genes (FIG. 1). From the 756 toxin gene positive isolates 635 (84%) were identified as Type A (a toxin only), however β, ε and ι, toxins were also detected in the clostridia silage isolates.

Genetic RAPD fingerprint patterns displayed diversity among the 590 feed isolates that successfully amplified. These isolates were obtained from 70 different farm's samples and formed 133 clusters based on 75% similarity according to the Dice correlation method. The largest cluster was 40 isolates (9.7%) (FIG. 3).

Out of the 3,958 isolates collected 3,202 isolates (80.9%) were found to be non-toxigenic clostridia. Sequencing representatives (n=345) from the non-toxigenic Clostridia displayed two dominate clostridia groups: C. bifermentans (Paraclostridium bifermentans and P. benzoelyticum) and C. beijerinckii group (C. diolis, C. beijerinckii, C. chromiireducens, C. saccharoperbutylacetonicum, C. puniceum, and C. saccharobutylicum). C. beijerinckii species are known producers of acetone and butanol. C. bifermentans species are rare opportunistic pathogens that can produce 1, 3-propanediol. These two main identification types of the non-toxigenic clostridia group made up 44% of the non-toxigenic isolates (FIG. 2.).

Representatives (n=196) from each individual farm's RAPD dendrogram were selected to capture the diversity of the C. perfringens population and subjected to inhibition assays. The combined data of the individual locations is made up of 41 different sites (Table 3). Antimicrobial testing using the bacteriocin turbidity assay displayed good inhibition of most feed C. perfringens isolates using bacteriocin harvested from 747, 1104, 1541, 1781, and 2018. The bacteriocin from at least one of the strains 747, 1104, 1541, 1781, and 2018 were able to inhibit the growth >60% of 126 (64.3%) of the 196 isolates tested. At least one bacteriocin strain was able to inhibit the growth of 97 (49.5%) of the 196 isolates tested by greater than 79%. Strain 747 had the highest overall inhibition across all C. perfringens silage isolates tested at 54.9%.

Non-toxigenic clostridia isolates (n=14) collected from two different dairies in Texas were also subjected to inhibition assays. The isolates tested consisted of C. bifermentans group, C. butyricum, C. beijerinckii group, C. ghonii, Clostridium tertium group, and C. sordellii (Table 4). The bacteriocin from at least one of the strains 747, 1104, 1541, 1781, 1999 and 2018 were able to inhibit the growth >60% of 11 (78.6%) of the 14 isolates tested. At least one bacteriocin strain was able to inhibit the growth of 9(64.3%) of the 14 isolates tested by greater than 79%. Strain 747 had the highest overall inhibition across all non-toxigenic clostridia silage isolates tested at 77.2%.

TABLE 2 Number of samples and average clostridia count results by location Average Clostridia Location Sample # (CFU/g) 1 4 1.0E+01 2 1 4.0E+01 3 2 2.3E+01 4 6 1.2E+03 5 18 8.7E+01 6 34 1.6E+02 7 2 3.5E+01 8 9 5.6E+02 9 26 6.8E+01 10 26 5.0E+01 11 24 7.9E+01 12 15 3.3E+03 13 4 4.9E+02 14 2 3.0E+01 15 14 5.7E+01 16 6 4.7E+01 17 1 9.5E+01 18 1 2.5E+01 19 1 1.0E+01 20 1 5.0E+01 21 12 3.1E+01 22 6 2.9E+01 23 2 1.0E+01 24 19 3.1E+01 25 2 5.1E+02 26 31 5.5E+03 27 3 6.7E+00 28 1 3.0E+01 29 5 1.1E+02 30 9 7.2E+02 31 4 2.3E+02 32 33 3.6E+01 33 18 1.9E+02 34 1 1.5E+01 35 5 1.9E+03 36 2 1.0E+01 37 13 7.7E+01 38 6 9.2E+00 39 17 3.4E+02 40 10 9.0E+01 41 47 4.0E+02 42 11 1.2E+02 43 9 5.3E+01 44 13 6.1E+03 45 3 3.7E+01 46 4 2.0E+02 47 4 1.4E+01 48 12 3.4E+05 49 5 9.0E+00 50 12 2.9E+01 51 20 2.0E+01 52 34 1.1E+02 53 14 6.4E+02 54 2 1.0E+01 55 1 0.0E+00 56 9 3.4E+03 57 5 9.2E+02 58 10 2.7E+01 59 2 1.0E+01 60 13 7.5E+01 61 6 2.7E+03 62 2 1.9E+02 63 8 2.0E+02 64 2 7.5E+00 65 7 1.0E+02 66 13 6.0E+01 67 21 2.3E+02 68 21 7.6E+03 69 2 1.5E+01 70 37 9.7E+02 71 17 3.0E+02 72 4 9.5E+01 73 7 1.6E+02 74 3 1.1E+02 75 10 5.5E+01 76 11 7.1E+01 77 4 4.0E+02 78 5 9.6E+01 79 9 3.3E+01 80 9 1.2E+02 81 47 8.9E+01 82 14 5.1E+01 83 8 1.7E+04 84 2 7.5E+00 85 1 5.0E+01 86 9 4.2E+01 87 7 3.3E+01 88 13 3.3E+01 89 4 1.5E+01 90 13 1.2E+02 91 14 1.3E+02 92 7 2.6E+01 93 7 2.4E+04 94 12 1.1E+04 95 3 1.2E+01 96 2 1.0E+01 97 2 4.8E+01 98 3 2.2E+01 99 1 1.0E+03 100 3 8.3E+00 101 7 8.6E+00 102 10 2.5E+04 103 2 8.5E+01 104 44 7.5E+02 105 17 9.5E+01 106 1 3.0E+01 107 12 7.0E+01 108 17 1.3E+02 109 46 3.2E+01 110 14 3.0E+01 111 3 8.3E+00 Total Samples 1,169 Average 4.2E+03 Clostridial level

TABLE 3 Percent inhibition of each silage C. perfringens isolate tested against bacteriocin harvested from Bacillus isolates. Bacillus Strains Isolate ID Location Sample Type 747 1104 1541 1781 2018 S72.11.1 Location 4 Dry Alfalfa 8.0% 2.6% 0.0% 5.3% 20.3% S72.11.4 Location 4 Dry Alfalfa 0.0% 0.0% 99.3% 0.0% 3.6% S72.18.7 Location 4 Pellets 88.2% 99.8% 99.7% 98.3% 61.2% S72.4.6 Location 4 Deep Silage 58.6% 79.8% 79.2% 58.0% 69.5% S114.3.5 Location 5 TMR Pen 3 99.2% 73.6% 79.1% 87.6% 87.2% S114.7.3 Location 5 Dry Cow TMR 99.6% 99.8% 99.8% 99.5% 99.5% S120.1.8 Location 9 Dry Cow TMR 100.2% 5.6% 99.9% 12.6% 43.6% S120.3.1 Location 9 1st Calf Heifer TMR 99.0% 25.6% 100.0% 23.0% 25.9% S120.3.10 Location 9 1st Calf Heifer TMR 0.0% 0.0% 0.0% 0.0% 0.0% S120.3.3 Location 9 1st Calf Heifer TMR 18.8% 0.0% 0.0% 0.0% 0.0% S120.4.2 Location 9 Fresh Cow TMR 100.0% 97.5% 100.0% 10.6% 81.0% S120.4.6 Location 9 Fresh Cow TMR 76.5% 75.1% 77.0% 75.3% 69.7% S120.5.6 Location 9 Lac. Cow TMR P5 26.9% 0.0% 0.0% 0.0% 0.0% S120.5.7 Location 9 Lac. Cow TMR P10 0.0% 0.0% 0.0% 0.0% 0.0% S120.7.1 Location 9 Ground Corn 0.0% 0.0% 0.0% 0.0% 0.0% S97.16.2 Location 10 Grass Hay 74.6% 74.1% 73.7% 74.6% 74.6% S50.4.1 Location 14 Fresh Cow TMR 0.0% 0.0% 0.0% 0.0% 0.0% S50.4.6 Location 14 Fresh Cow TMR 0.0% 0.0% 0.0% 0.0% 0.0% S50.5.3 Location 14 Prefresh TMR 0.0% 0.0% 0.0% 0.0% 0.0% S50.5.5 Location 14 Prefresh TMR 0.0% 0.0% 0.0% 0.0% 0.0% S50.5.8 Location 14 Prefresh TMR 0.0% 0.0% 0.0% 10.4% 13.9% S50.6.1 Location 14 High Cow TMR 0.0% 0.0% 0.0% 0.0% 0.0% S50.6.3 Location 14 High Cow TMR 0.0% 0.0% 0.0% 0.0% 0.0% S53.11.1 Location 17 Fresh 0.0% 0.0% 0.0% 0.0% 0.0% S53.5.5 Location 17 Dry Cows 0.0% 0.0% 0.0% 0.0% 2.1% S53.5.6 Location 17 Dry Cows 32.6% 35.9% 9.1% 41.1% 40.7% S53.5.7 Location 17 Dry Cows 0.0% 0.0% 0.0% 0.0% 0.0% S53.7.3 Location 17 Ground Corn 0.0% 6.4% 0.0% 7.3% 0.0% S53.7.6 Location 17 Ground Corn 0.0% 0.0% 0.0% 0.0% 0.0% S53.7.7 Location 17 Ground Corn 10.9% 0.0% 0.0% 0.0% 8.4% 30.4.2 Location 19 Corn Silage B7 43.0% 43.0% 46.0% 45.0% 43.0% S54.1.1 Location 20 Wet Brewers Grain 89.8% 91.3% 93.1% 0.0% 2.6% S54.16.2 Location 20 Fresh Cow TMR 0.0% 0.0% 0.0% 0.0% 0.0% S54.18.3 Location 20 High Cow TMR 0.0% 0.0% 0.0% 0.0% 0.0% S54.19.4 Location 20 Far Off Dry TMR 0.0% 0.0% 0.0% 0.0% 0.0% S70.1.6 Location 22 Corn Silage 99.6% 99.7% 81.4% 99.7% 99.6% S70.1.7 Location 22 Corn Silage 99.7% 99.7% 100.0% 99.7% 99.7% S70.10.9 Location 22 High Cow Premix 99.8% 100.0% 100.0% 100.0% 100.0% S117.2.6 Location 25 Canola 97.0% 38.5% 21.6% 0.0% 27.6% S117.4.10 Location 25 Cotton Seed 67.4% 27.1% 5.1% 17.9% 22.6% S41.10.1 Location 27 Protein Mix 98.9% 98.8% 97.1% 98.7% 98.7% S41.12.2 Location 27 Molasses 84.2% 78.8% 83.4% 84.9% 82.8% S63.1.1 Location 27 Corn Silage B1 2015 98.6% 96.1% 98.9% 99.2% 99.2% S63.2.2 Location 27 Alfalfa Haylage B3 7.7% 67.1% 23.4% 41.9% 50.0% 2015 S63.2.4 Location 27 Alfalfa Haylage B3 89.0% 74.3% 89.6% 88.7% 87.8% 2015 S63.3.1 Location 27 Alfalfa Haylage B4 98.1% 54.4% 98.1% 37.5% 98.1% 2nd 2016 S63.5.10 Location 27 TMR 5 71.9% 9.1% 59.8% 56.5% 63.6% S63.5.2 Location 27 TMR 5 92.2% 56.7% 68.8% 82.0% 67.1% S63.5.3 Location 27 TMR 5 46.8% 65.5% 53.4% 66.5% 61.8% S63.5.4 Location 27 TMR 5 75.8% 10.5% 53.8% 50.3% 35.7% S63.5.5 Location 27 TMR 5 82.9% 15.9% 89.9% 87.7% 86.4% S63.5.6 Location 27 TMR 5 99.5% 99.8% 94.7% 99.3% 99.1% S63.5.7 Location 27 TMR 5 96.4% 94.6% 79.8% 97.3% 82.0% S63.5.9 Location 27 TMR 5 98.0% 26.5% 93.4% 93.9% 79.8% S63.6.1 Location 27 Dry Corn 98.8% 20.6% 95.6% 98.1% 95.6% S63.6.3 Location 27 Dry Corn 32.0% 43.1% 45.5% 0.0% 52.2% S63.6.5 Location 27 Dry Corn 99.5% 20.1% 96.4% 62.3% 99.3% S63.9.1 Location 27 Protein Blend with 3.6% 36.1% 86.8% 53.6% 80.0% Bacillus S73.8.1 Location 28 Haylage Deep 24.9% 0.0% 0.0% 25.5% 0.0% S73.8.2 Location 28 Haylage Deep 18.3% 0.0% 0.0% 23.7% 0.0% S59.10.2 Location 33 High Cow Ration Pen 33.9% 33.9% 28.8% 45.7% 24.2% 13 S59.10.3 Location 33 High Cow Ration Pen 44.0% 44.9% 44.7% 45.0% 45.7% 13 S59.11.1 Location 33 Dry Cow Ration 87.0% 71.3% 62.5% 76.0% 68.5% S59.14.1 Location 33 Canola 0.0% 0.0% 0.0% 0.0% 0.0% S59.14.4 Location 33 Canola 65.5% 37.8% 33.9% 75.6% 33.1% S59.14.7 Location 33 Canola 35.1% 0.0% 0.0% 7.9% 0.0% S59.15.2 Location 33 One Track 3.4% 2.6% 0.0% 5.5% 5.0% S59.8.1 Location 33 Close Up Cow Pen 8 12.7% 5.3% 4.8% 6.7% 0.0% S59.8.2 Location 33 Close Up Cow Pen 8 17.4% 22.2% 18.7% 14.0% 22.1% S59.8.3 Location 33 Close Up Cow Pen 8 10.1% 17.2% 13.9% 46.5% 12.6% S59.9.1 Location 33 Medium Cow Ration 95.5% 61.9% 58.7% 100.0% 43.9% Pen 4 S59.9.2 Location 33 Medium Cow Ration 83.9% 62.1% 44.0% 72.5% 64.1% Pen 4 S69.5.5 Location 34 Close Up TMR 100.0% 100.0% 100.0% 100.0% 100.0% S69.6.2 Location 34 Dry Cow TMR 6.2% 0.0% 4.7% 4.8% 9.3% S69.6.6 Location 34 Dry Cow TMR 28.0% 0.0% 2.7% 19.6% 30.9% S57.6.5 Location 36 Cow Ration Lac 0.0% 0.0% 0.0% 0.0% 0.0% S58.4.1 Location 36 Early Lac Ration 34.9% 57.8% 34.1% 96.2% 37.9% S58.4.10 Location 36 Early Lac Ration 100.0% 100.0% 100.0% 100.0% 100.0% S58.4.2 Location 36 Early Lac Ration 50.1% 15.2% 24.7% 86.1% 15.3% S58.4.9 Location 36 Early Lac Ration 15.9% 0.0% 0.0% 0.0% 100.0% S58.5.1 Location 36 Mid Lac Ration 8.5% 14.3% 9.0% 9.8% 5.6% S58.5.2 Location 36 Mid Lac Ration 68.9% 47.2% 44.9% 73.7% 37.3% S58.6.1 Location 36 Dry Cow Ration 6.1% 0.0% 0.0% 3.4% 100.0% S58.8.1 Location 36 Dry Cow Ration 95.6% 55.1% 100.0% 100.0% 0.0% S58.8.3 Location 36 Pellets 88.1% 59.2% 55.7% 94.9% 80.8% S58.9.4 Location 36 Cotton 49.3% 38.6% 14.5% 100.0% 22.8% S58.9.5 Location 36 Cotton 78.2% 40.6% 45.5% 77.9% 49.0% 21.2.3 Location 39 Haylage B3 100.0% 98.0% 74.0% 99.0% 97.0% 21.2.4 Location 39 Haylage 33.9% 5.2% 4.7% 2.2% 5.3% 21.2.5 Location 39 Haylage B3 98.0% 49.0% 42.0% 94.0% 48.0% 21.2.7 Location 39 Haylage B3 98.0% 96.0% 96.0% 98.0% 96.0% S51.2.2 Location 43 Haylage 1.9% 0.0% 0.0% 0.0% 0.0% S104.4.4 Location 44 Alfalfa Haylage 4th 0.0% 0.0% 0.0% 0.0% 95.2% S104.4.7 Location 44 Alfalfa Haylage 4th 0.0% 0.0% 0.0% 0.0% 0.0% CSS-4 Location 44 Corn Silage 29.5% 31.7% 96.2% 38.3% 28.6% HSS-1 Location 44 Haylage 99.9% 100.0% 99.9% 100.0% 99.9% 28.1.2 Location 44 Ryelage 64.0% 63.0% 91.0% 63.0% 69.0% S74.7.1 Location 45 Ground Corn 73.9% 65.9% 46.1% 35.3% 65.9% S76.3.5 Location 46 Baylage 4th Home 77.5% 36.4% 100.0% 37.2% 73.6% S76.3.8 Location 46 Baylage 4th Home 50.7% 14.2% 97.8% 51.6% 42.7% S75.3.1 Location 47 Close-up TMR 100.0% 99.2% 100.0% 100.0% 100.0% S75.3.10 Location 47 Close-up TMR 100.0% 100.0% 100.0% 100.0% 100.0% S75.5.1 Location 47 Late TMR 34.4% 24.2% 40.7% 60.0% 43.1% S75.8.1 Location 47 Calf Pellets 100.0% 100.0% 100.0% 100.0% 100.0% S75.8.3 Location 47 Calf Pellets 100.0% 99.8% 99.7% 100.0% 100.0% S75.8.5 Location 47 Calf Pellets 100.0% 100.0% 100.0% 100.0% 99.2% S75.8.9 Location 47 Calf Pellets 100.0% 100.0% 100.0% 100.0% 100.0% S75.9.1 Location 47 Soy Hulls 100.0% 100.0% 99.7% 100.0% 99.7% S75.9.2 Location 47 Soy Hulls 99.7% 100.0% 99.5% 99.8% 99.8% S61.10.6 Location 52 TMR 1 44.5% 20.2% 6.8% 54.3% 16.4% S61.12.1 Location 52 TMR 3 57.1% 0.0% 40.8% 12.8% 9.3% S61.12.10 Location 52 TMR 3 0.0% 0.0% 0.0% 0.0% 0.0% S61.12.5 Location 52 TMR 3 0.0% 0.0% 0.0% 0.0% 0.0% S61.12.7 Location 52 TMR 3 0.0% 0.0% 0.0% 0.0% 0.0% S61.3.2 Location 52 Corn Silage Face 95.0% 92.8% 92.3% 90.3% 89.9% S61.3.4 Location 52 Corn Silage Face 0.0% 94.4% 94.4% 0.0% 92.1% S61.5.3 Location 52 Dry Hay Ontario 95.3% 7.7% 98.3% 89.8% 95.1% S99.6.5 Location 53 Close Up TMR 0.0% 1.3% 16.7% 0.0% 0.0% Silage 3-3 Location 54 Silage 98.9% 99.6% 100.0% 99.3% 99.8% Silage 3-4 Location 54 Silage 98.4% 100.0% 100.0% 98.8% 100.0% Silage 3-5 Location 54 Silage 34.1% 0.0% 29.8% 3.8% 30.8% Silage 8-1 Location 54 Silage 86.7% 100.0% 86.7% 86.7% 100.0% S36.1.6 Location 55 Face Wheatlage 100.0% 100.0% 100.0% 100.0% 100.0% S36.1.7 Location 55 Face Wheatlage 100.0% 0.0% 100.0% 0.0% 100.0% S81.10.6 Location 59 Wheat Midds 100.0% 100.0% 100.0% 100.0% 100.0% S81.11.1 Location 59 Bird Seed Screening 91.5% 82.3% 91.1% 83.0% 80.1% S81.11.7 Location 59 Bird Seed Screening 30.5% 32.9% 22.6% 37.2% 27.1% S81.3.6 Location 59 3rd Haylage 25.4% 0.0% 0.0% 0.0% 0.0% S81.7.4 Location 59 Cow TMR 94.5% 90.9% 89.5% 92.4% 92.0% S89.15.3 Location 60 WOM #1046 2.8% 0.0% 0.0% 8.3% 30.0% S89.20.7 Location 60 WOM #1550 11.3% 0.0% 0.0% 21.0% 24.7% S89.20.9 Location 60 WOM #1550 0.0% 0.0% 0.0% 0.0% 28.8% S89.3.1 Location 60 Ryegrass Haylage 26.9% 4.3% 0.0% 13.1% 5.0% S49.10.4 Location 60 Pen 432 Conventional 0.0% 0.0% 0.0% 0.0% 0.0% 30 Ration S49.5.3 Location 60 Pen 146 Natural 22.4% 12.7% 19.5% 2.6% 18.1% Starter S71.10.10 Location 63 Dry Cow Ration 59.7% 66.2% 44.7% 10.4% 36.8% S71.17.1 Location 63 Burrs 0.7% 0.0% 10.5% 14.7% 34.2% S71.17.2 Location 63 Burrs 0.0% 0.0% 8.7% 1.9% 19.6% S71.9.3 Location 63 Late Lac Ration 79.5% 34.5% 20.3% 49.5% 39.8% S71.9.5 Location 63 Late Lac Ration 65.7% 63.5% 64.9% 69.5% 54.8% 3.1.3 Location 65 Corn Silage 100.0% 100.0% 100.0% 100.0% 100.0% 3.1.4 Location 65 Corn Silage 72.0% 68.0% 91.0% 70.0% 80.0% 31.2.1 Location 65 Haylage 43.0% 37.0% 36.0% 41.0% 41.0% 27.2.11 Location 67 Haylage 98.0% 91.0% 81.0% 95.0% 91.0% 27.3.2 Location 67 Oatlage 93.0% 66.0% 66.0% 80.0% 75.0% 27.3.3 Location 67 Oatlage 100.0% 100.0% 100.0% 100.0% 100.0% S91.2.4 Location 68 Grass Haylage 82.4% 77.4% 80.5% 86.6% 82.2% S91.2.5 Location 68 Grass Haylage 77.9% 50.5% 57.2% 74.1% 40.4% S91.3.5 Location 68 Pre Fresh 65.5% 73.5% 76.1% 64.5% 66.7% S91.5.4 Location 68 Low TMR 73.8% 76.3% 74.9% 62.4% 66.0% S56.1.2 Location 72 Pellets 98.1% 96.7% 97.5% 96.6% 96.2% S56.1.3 Location 72 Pellets 11.8% 1.9% 5.5% 0.0% 4.5% S56.7.6 Location 72 Corn Silage Face 38.8% 4.5% 0.0% 13.2% 32.4% S56.9.1 Location 72 Milk Cow TMR 40.6% 39.7% 56.7% 39.8% 54.8% S56.9.3 Location 72 Milk Cow TMR 4.6% 0.0% 6.1% 0.0% 11.5% S56.9.6 Location 72 Milk Cow TMR 0.0% 0.0% 0.0% 0.0% 0.0% S56.9.7 Location 72 Milk Cow TMR 0.0% 0.0% 0.0% 0.0% 0.0% S52.14.1 Location 76 Robot Barn 0.0% 0.0% 11.8% 20.6% 69.0% S52.14.7 Location 76 Robot Barn 0.0% 0.0% 0.0% 0.0% 0.0% S57.4.1 Location 80 New Alfalfa Haylage 0.0% 0.0% 0.0% 0.0% 0.0% Compost RF-1 Location 85 Corn Silage 100.0% 100.0% 100.0% 100.0% 100.0% ROL-1 Location 85 Haylage-Old 92.6% 94.7% 94.5% 94.4% 89.5% ROL-3 Location 85 Haylage-Old 99.9% 100.0% 99.8% 99.9% 99.7% ROS-1 Location 85 Haylage-Old 35.9% 16.1% 17.4% 17.0% 18.5% ROS-3 Location 85 Haylage-Old 100.0% 20.3% 28.9% 25.1% 50.5% S102.1.10 Location 85 Baylage Mark 58.5% 62.1% 66.4% 57.4% 57.3% S102.1.3 Location 85 Baylage Mark 35.8% 44.2% 45.8% 29.2% 41.2% S102.1.9 Location 85 Baylage Mark 49.2% 51.3% 43.4% 50.9% 53.4% S102.11.1 Location 85 Fresh TMR 0.0% 11.7% 0.0% 0.0% 0.0% S102.7.3 Location 85 Fresh Heifer TMR 80.3% 1.3% 63.3% 74.4% 76.7% S102.8.4 Location 85 Close Up Cow TMR 67.8% 49.4% 85.5% 71.1% 65.3% S102.8.5 Location 85 Close Up Cow TMR 59.2% 40.0% 71.6% 62.4% 55.8% S102.8.8 Location 85 Close Up Cow TMR 80.5% 59.8% 80.0% 85.2% 85.4% S111.4.9 Location 86 Lactation 1 TMR 75.7% 73.4% 99.5% 80.1% 92.3% 20.1.3 Location 87 Corn Silage B1 12.0% 12.0% 11.0% 17.0% 12.0% 20.1.5 Location 87 Corn Silage B1 21.0% 18.0% 37.0% 25.0% 19.0% 20.3.1 Location 87 Haylage B6 85.0% 89.0% 84.0% 90.0% 80.0% 20.3.3 Location 87 Haylage 90.0% 92.7% 94.1% 93.1% 93.0% 20.3.5 Location 87 Haylage 99.7% 100.0% 99.9% 99.9% 100.0% 20.4.6 Location 87 Corn Silage B7 100.0% 100.0% 100.0% 100.0% 100.0% S80.4.1 Location 91 Cow TMR 0.0% 0.0% 0.0% 33.7% 13.4% S55.9.1 Location 92 Dry Cow TMR 30.4% 5.7% 0.0% 19.2% 0.0% S40.1.8 Location 99 Oatlage 98.4% 86.8% 97.0% 95.8% 93.5% S47.1.6 Location 99 Alfalfa Haylage West 19.3% 0.0% 5.9% 0.0% 28.6% S60.4.1 Location 99 Ryelage 96.9% 50.5% 96.9% 97.5% 96.9% S60.4.10 Location 99 Ryelage 95.4% 94.8% 95.4% 96.1% 94.8% S60.4.4 Location 99 Ryelage 99.0% 99.1% 99.0% 98.8% 99.0% S60.4.6 Location 99 Ryelage 99.4% 94.6% 99.5% 99.4% 99.5% S60.4.8 Location 99 Ryelage 97.9% 19.5% 97.9% 98.0% 98.5% S60.5.1 Location 99 Milk Cow TMR 97.6% 27.6% 93.3% 89.8% 83.5% S60.7.1 Location 99 Whey 98.8% 99.3% 99.0% 98.8% 99.0% S60.7.10 Location 99 Whey 98.4% 10.1% 98.7% 97.6% 94.3% S60.7.3 Location 99 Whey 99.1% 97.9% 99.0% 98.5% 98.6% S60.7.8 Location 99 Whey 82.6% 85.5% 98.6% 98.6% 99.0% S60.7.9 Location 99 Whey 98.8% 96.2% 98.6% 98.2% 98.8% S77.6.3 Location 100 Heifer Haylage Deep 96.3% 96.3% 96.3% 97.9% 95.5% Average Percent 54.9% 43.4% 51.4% 50.14% 51.55% Inhibition

TABLE 4 Percent inhibition of non-toxigenic clostridia isolates collected from Texas feed samples against bacteriocin harvested from Bacillus isolates. Bacillus Strains Isolate ID 16S ID 747 1104 1541 1781 1999 2018 S35.1.1 C. butyricum 76.8% 51.8% 41.4% 43.8% 41.2% 76.3% S37.1.1 Clostridium tertium group 94.8% 0.0% 34.2% 29.3% Not Tested 84.0% S37.11.1 Clostridium sordellii 24.5% 23.1% 23.0% 23.6% Not Tested 24.2% group S37.11.2 Clostridium beijerinckii 24.3% 22.8% 21.8% 20.0% Not Tested 20.3% group S37.11.3 Clostridium beijerinckii 24.1% 21.9% 25.5% 21.1% Not Tested 28.9% group S37.11.4 Clostridium sordellii 97.3% 97.2% 96.5% 96.4% Not Tested 97.0% group S37.11.5 Clostridium tertium group 96.0% 36.6% 77.4% 80.0% Not Tested 80.7% S37.11.6 Clostridium sordellii 75.5% 0.0% 7.8% 74.8% Not Tested 76.2% group S37.11.7 Clostridium sartagoforme 89.8% 30.8% 47.9% 70.0% Not Tested 79.0% S37.11.8 Clostridium sartagoforme 94.1% 0.0% 33.9% 0.0% Not Tested 57.4% S37.11.9 C. beijerinckii 96.9% 45.7% 62.0% 39.3% 36.9% 66.2% S37.12.3 Clostridium sordellii 97.2% 49.4% 96.0% 96.3% Not Tested 86.9% group S37.12.4 Clostridium sordellii 93.5% 0.0% 0.0% 15.3% Not Tested 27.6% group S37.12.5 C. bifermentans group 96.0% 70.0% 68.5% 69.3% 74.8% 69.5% Average Percent Inhibition 77.2% 32.1% 45.4% 48.5% 51.0% 62.4%

Conclusion:

Feed samples (1,169) were collected from 111 different locations and tested for levels of clostridia. These levels ranged from <10 to 4.1 E+06 CFU/g, with an average enumeration count of 4.2 E+03 CFU/g across all samples. All locations, except for one site, where only one sample was collected, had detectable levels of clostridia.

Of the 3,958 presumptive clostridia isolates collected from 100 different locations, 756 tested positive for at least one C. perfringens toxin gene. The majority (84%) of these positive isolates were identified as Type A. Genetic analysis by RAPD PCR of 590 of these toxigenic clostridia isolates showed a diverse population consisting of 133 clusters. For the most part, isolates did not seem to group based on location, except for a few smaller clusters. There were 38 isolates that did not cluster with any other isolates.

Representative isolates (345) from the non-toxigenic isolates (3202) were identified by sequencing of the 16S rRNA gene. C. beijerinckii group (23%) and C. bifermentans group (21%) were predominant. Other non-toxigenic species identified included C. tertium group, C. butyricum, C. sordellii group, C. perfringens, Terrisporobacter species, C. sardiniense group and Romboutsia lituserburensis.

Antimicrobial screening against both toxigenic and non-toxigenic clostridia showed inhibition by the inventors' identified Bacillus strains. One or more of the Bacillus strains 747, 1104, 1541, 1781, and 2018 inhibited 126 of the 196 C. perfringens isolates. For the non-toxigenic isolates, 11 of the 14, were inhibited by one or more of the Bacillus strains 747, 1104, 1541, 1781, 1999, and 2018.

Example 2: The Effect of a Combination of Bacillus Strains on the Ensiling of Alfalfa in Mini-Silos

Introduction:

Clostridia fermentation in silage is controlled using lactic acid bacteria as silage inoculants to support the preservation of the crop and by ensuring the crop is harvested and ensiled under low-moisture conditions. However, managing the on-farm conditions such as weather that would impact the moisture content of the crop at harvest is not always practical or possible, and often ensiling occurs under sub-optimal conditions. Although bacilli are considered silage spoilage organisms, members of the Bacillus genera are known to produce antimicrobial compounds capable of inhibiting competing bacteria in the surrounding environment, and have demonstrated efficacy in controlling the growth of clostridia. The purpose of this study was to determine the efficacy of Bacillus strains, in accordance with this embodiment of the present invention, at controlling the growth of clostridia spoilage organisms in alfalfa ensiled in mini-silos.

Materials and Methods:

Two of the Bacillus strains, in accordance with this embodiment of the present invention, Bacillus 1104 and 1781, were combined in equal proportions as a silage inoculant and applied to forage to test the effect of a Bacillus-based silage inoculant on the physical and chemical properties of the forage material, as well as its ability to suppress secondary fermentation associated with clostridia growth.

Freshly cut alfalfa haylage was treated with the following silage inoculant treatments:

1) Control without silage inoculant;

2) Low Dose Bacillus inoculant;

3) High Dose Bacillus inoculant

4) Lactic acid bacteria (LAB) inoculant

5) High Dose Bacillus inoculant+LAB inoculant

6) Mega Dose Bacillus inoculant+LAB inoculant

The Bacillus inoculant contained 50% strains 1104 1781. The LAB inoculant contained 30% Lp115 (Lactobacillus plantarum), 30% Pj300 (Pediococcus acidilactici), 30% P751 (P. pentosaceus), and 10% Enterococcus faecium. Inoculant target application rates per gram of silage were: Low Dose Bacillus at 5,000 CFU/g, High Dose Bacillus at 50,000 CFU/g, LAB at 150,000 CFU/g, High Dose Bacillus+LAB at a combination of respective Bacillus and LAB treatment rates, Mega Dose Bacillus+LAB at 5,000,000 CFU/g Bacillus and the respective LAB rate (Table 5).

TABLE 5 Inoculant Doses of Six Treatments Applied to Alfalfa Forage Material Bacillus Dose, LAB Dose, Treatment CFU/g CFU/g Control 0 0 Low Dose Bacillus 5,000 0 High Dose Bacillus 50,000 0 LAB 0 150,000 High Dose Bacillus + LAB 50,000 150,000 Mega Dose Bacillus + LAB 5,000,000 150,000

Each treatment was applied to 1,000 g of forage material and packed in 8 oz glass ball jars with sealed lids at a density of 39 lb/ft³. Jars were stored at room temperature and enumerations were performed on days 7, 30, 78, 90, and 182 for bacteria and microbes of interest (Bacillus, LAB, Escherichia coli and coliforms, clostridia, yeast and mold). The untreated sample (control) was tested on Day 0 for pH and initial background bacteria present. At each time point, pH readings were also recorded for each treated sample and analyzed for presence and concentration of volatile fatty acids (VFAs) on an as-sampled basis. The following VFAs were evaluated: lactic acid, acetic acid, butyric acid, isobutyric acid, propionic acid, valeric acid, and isovaleric acid. Final VFA results were reported as the average of duplicate values.

Results and Discussion:

The pH levels for all six treatments dropped from 5.70 on Day 0 to 4.19-4.33 by Day 30 (FIG. 4). From Day 30 to Day 182, the pH values for all treatments remained mostly stable with the Control maintaining the highest pH level over this time compared to samples treated with inoculant. This data suggests that the addition of Bacillus in applied silage inoculants can help lower the pH rapidly, starting the fermentation process sooner, and can help maintain a more stable pH during the stable phase (or storage phase) of the ensiling process. A lower pH can help inhibit spoilage bacteria, improve the digestibility of the feed, reduces proteolytic enzymes, inhibits anaerobic bacteria, shifts fermentation end products, and increases hydrolysis of hemicellulose

E. coli population in the control and the other five treatments dropped from 43 CFU/g on Day 0 to 10 CFU/g on Day 78 (Table 6). From Day 78 to Day 182, E. coli levels for the control and all treatments stayed consistently at 10 CFU/g. Coliform counts were maintained at 10 CFU/g for both the control sample and other four treatment samples from Day 78 to Day 182. The addition of Bacillus in the applied silage inoculant treatments did not result in the E. coli or coliform population levels increasing over time during the ensiling process.

TABLE 6 E. coli and coliform population changes over time per treatment E. coli Coliforms Day Treatment (CFU/g) (CFU/g) 0 Control 43 N/A 78 Control 10 10 78 Bacillus Low Dose 10 10 78 Bacillus High Dose 10 10 78 LAB 10 10 78 High Dose + LAB 10 10 90 Control 10 10 90 Bacillus Low Dose 10 10 90 Bacillus High Dose 10 10 90 LAB 10 10 90 High Dose + LAB 10 10 90 Mega Dose + LAB 10 10 182 Control 10 10 182 Bacillus Low Dose 10 10 182 Bacillus High Dose 10 10 182 LAB 10 10 182 High Dose + LAB 10 10 182 Mega Dose + LAB 10 10

As expected, treatments without Bacillus added (Control and LAB) had the lowest Bacillus counts over the time course of measurements compared to treatments with added Bacillus (FIG. 5). Generally, Bacillus levels in the Control and LAB treatment were lower than the other five treatments at all time points. The Mega Dose Bacillus+LAB Treatment had the highest level of Bacillus growth throughout the trial compared to all other treatments. By trial end, all samples treated with inoculant had higher levels of Bacillus detected in them than in the Control, indicating that the Bacillus strains present in the silage inoculant remained stable and experienced little to no die off throughout the ensiling process. LAB populations were reduced by 2-3 logs after 60 d post ensiling for all treatments, indicating a normal lactic acid bacterial fermentation to preserve the forage (FIG. 6).

Yeast counts decreased from Day 0 to Day 30 for all treatments and the control (FIG. 7). After Day 30 all treatments, with the exception of the Bacillus High Dose+LAB treatment on Day 78, had consistent yeast counts of 10 CFU/g and stayed at this concentration throughout the remainder of the experiment, except for the Control. The Control had the highest levels of yeast growth among the treatments tested by the end of the trial on Day 180. This data indicates that the addition of Bacillus did not negatively affect the ability of the lactic acid bacteria inoculant to inhibit yeast spoilage organism growth during silage preservation.

Mold counts decreased from Day 0 to Day 30 for all treatments and the Control (FIG. 8). After Day 30 all treatments had consistent mold counts of 10 CFU/g for the remainder of the time points tested, whereas the Control treatment had a mold count of 10 CFU/g from Day 30 to Day 90 and then increased to 3.5 CFU/g for the last plating on Day 182. These data indicate that the addition of Bacillus did not negatively affect the ability of the lactic acid bacteria inoculant to reduce and control mold spoilage organism growth.

All the inoculant treatments with the exception of the Bacillus High Dose+LAB, had similar or lower clostridia levels by the end of the experiment compared to the Control (FIG. 9). The Bacillus Low Dose had the lowest levels of clostridia for the last two time points tested out of all of the treatments. This data indicates that Bacillus by itself and/or in combination with LAB is controlling clostridia growth and secondary fermentation more effectively than when no treatment is administered.

Lactic acid concentration increased in all treatments from Day 0 to Day 78 and then decreased from Day 78 to Day 90 (FIG. 10). The control showed consistently lower levels of lactic acid at each time point compared to samples treated with the microbial inoculants. The highest levels of lactic acid were observed in the High Dose Bacillus+LAB treatment compared to all other treatments for each time point tested. Acetic acid concentrations increased in all treatment samples from Day 0 to Day 78 and then decreased from Day 78 to Day 90 (FIG. 11). For each time point tested, the High Dose Bacillus+LAB treatment had the highest concentration of acetic acid compared to all of the other treatments. The LAB treatment had the lowest concentration of acetic acid for all time points, except for Day 30 where the Mega Dose treatment was slightly lower. Concentrations of butyric acid, isobutyric acid, propionic acid, valeric acid, and isovaleric acid were not detected in any of the treatment samples at any of the time points measured.

Conclusions:

The pH dropped drastically within the first 30 days from a starting point of 5.7. From this point on, the pH readings were stable at this range until the end of the trial. All samples treated with Bacillus silage inoculant had lower pH compared to the controls. Lactic acid bacteria increased within the first few days of ensiling and then generally decreased over the course of the experiments, while Bacillus levels for all treated samples were higher than that of the controls. Both yeast and mold counts dropped drastically within the first 30 day of ensiling and then evened out until trial end. Treated samples had counts that were ten times lower than that of the control. Treated samples, except for the Bacillus High Dose+LAB treatment, had slightly lower levels of clostridia by trial end.

This data indicates that silage preservation is not negatively affected by the addition of these Bacillus strains as a silage inoculant. In fact, the Bacillus by itself and/or in combination with LAB is controlling clostridia growth and secondary fermentation, as well as yeast and mold growth, more effectively than when no treatment is administered.

Example 3: The Effect of a Combination of Bacillus Strains as Silage Inoculants on Alfalfa Silage on Farm and in Mini-Silos

Introduction:

Clostridia fermentation in silage is controlled using lactic acid bacteria as silage inoculants to support the preservation of the crop and by ensuring the crop is harvested and ensiled under low-moisture conditions. However, managing the on-farm conditions such as weather that would impact the moisture content of the crop at harvest is not always practical or possible, and often ensiling occurs under sub-optimal conditions. Although bacilli are considered silage spoilage organisms, members of the Bacillus genera are known to produce antimicrobial compounds capable of inhibiting competing bacteria in the surrounding environment, and have demonstrated efficacy in controlling the growth of clostridia. The purpose of this study was to determine the efficacy of Bacillus strains, in accordance with this embodiment of the present invention, at controlling the growth of clostridia spoilage organisms in alfalfa ensiled on farm and in mini-silos.

Materials and Methods:

Two of the Bacillus strains, in accordance with this embodiment of the present invention, Bacillus 1104 and 1781, were combined in equal proportions as a silage inoculant and applied to forage to test the effect of a Bacillus-based silage inoculant on the physical and chemical properties of the forage material, as well as its ability to suppress secondary fermentation associated with clostridia growth.

The study utilized alfalfa silage from second cutting and treatments consisted of a Control, silage inoculant treatment applied on the farm, silage inoculant treatment applied at a 1× dose in the laboratory, and silage inoculant treatment applied at a 10× dose in the laboratory. Both the farm and lab silage inoculant treatments contained 12.5% strains 1104 and 1781), 7% En. faecium, 22.5% each of LP 115 (L. plantarum), Pj300 (P. acidilactici), and P751 (P. pentosaceus).

Inoculant target application rate for the both the farm and laboratory applied silage inoculant treatments was 200,000 CFU/gram of crop, consisting of 150,000 CFU Lactic Acid bacteria (LAB) and 50,000 CFU Bacillus. The 10× dose included 2,000,000 CFU/gram of crop, consisting of 1,500,000 CFU LAB and 500,000 CFU Bacillus. Laboratory inoculant treatments were applied to 1,000 g of forage material and all treatments were packed in 8 oz glass ball jars with sealed lids at a density of 39 lb/ft³. Jars were stored at room temperature and enumerations were performed on days 8, 14, 28, 60, 90, and 176 days for bacteria of interest (Bacillus, LAB, E. coli and coliforms, clostridia, yeast and mold).

Initial background bacteria and pH was recorded for all treatments on Day 0. At each time point, pH readings were obtained for each treated sample and analyzed for presence and concentration of VFAs on an as-sampled basis. Volatile fatty acids, including lactic acid, acetic acid, butyric acid, isobutyric acid, propionic acid, valeric acid, and isovaleric acid, were processed in duplicate and measured. Final VFA results were reported as the average of the duplicate values.

Results and Discussion:

The pH decreased for all treatments in the initial measurement following treatment administration and was maintained over the course of the study (FIG. 12). The observed decrease in pH indicates active fermentation of the forage material and that long term silage preservation is not being negatively affected by the addition of Bacillus strains in a silage inoculant.

Except for the 10× silage inoculant dose, E. coli counts were reduced in inoculant treatment samples at a faster rate initially compared to the Control (FIG. 13). After Day 30, all treatments maintained a low level of E. coli (10 CFU/g) for the remainder of the experimental period. A similar reduction in coliform levels was observed for the inoculant treatment samples during the first 30 days of testing (FIG. 14). These data indicate that the treatments are controlling spoilage organism growth over the course of the fermentation process.

During the study, total Bacillus counts ranged between 6.7×10³ and 4.7×10⁵ CFU/g, with the highest Bacillus counts observed in the 10× silage inoculant dose treatment. After the Bacillus community was established in the treated samples after Day 60, Bacillus levels did not fluctuate much for the remainder of the trial. This shows that Bacillus is stable in feed samples and maintains activity under a wide range of conditions (FIG. 15). LAB counts increased initially to Day 30, and then decreased gradually over the remainder of the test period (FIG. 16).

During the first 30 days of the trial, the yeast population for all treatments and the Control were reduced and were maintained at low levels (10 CFU/g) over the course of the experimental period (FIG. 17). Mold counts responded similarly for all treatments through the trial period (FIG. 18). This data indicate that the fermentation process controlled yeast and mold spoilage in the silage and that practical application of the silage inoculant on the farm resulted in similar benefits as when the inoculant was applied under experimental conditions in the laboratory.

Clostridia counts were reduced in the first seven days of the trial for all treatments. (FIG. 19). All silage inoculant treatments had similar or lower counts of clostridia compared to the control treatment that was not administered a bacterial inoculant. This data indicates that the addition of silage inoculants to forage is controlling clostridia growth and secondary fermentation more effectively than when no treatment is administered.

Higher concentrations of lactic acid were observed in samples from all treatments at Day 14 and Day 90 compared to other sampling days (FIG. 20). The 10× silage inoculant dose had the highest levels of lactic acid the end of the trial period compared to the other treatments, indicating this treatment resulted in well preserved forage. A similar trend was observed for acetic acid concentrations, in which acetic acid levels increased by 1-3 fold from Day 0 to Day 90 (FIG. 21). Both the 10× silage inoculant dose administered in the laboratory and the silage inoculant administered on-farm had greater concentrations of acetic acid present at the end of the trial period compared to the untreated control, indicating that these treatments provided a more stable fermentation process for silage preservation.

Conclusions:

The pH decreased for all treatments in the initial measurement following treatment administration and was maintained over the course of the study. E. coli and coliform counts were reduced in inoculant treatment samples at a faster rate compared to the Control, except for the 10× silage inoculant dose. Treated samples had slightly lower levels of mold present compared to their untreated controls.

This data indicates that silage preservation is not negatively affected by the addition of these Bacillus strains to a silage inoculant. In fact, the Bacillus in combination with LAB improved the reduction of coliforms and prevented mold growth, more effectively than when no treatment was administered. The pH decreased for all treatments in the initial measurement following treatment administration and was maintained over the course of the study (FIG. 12). The observed decrease in pH indicates active fermentation of the forage material and that long term silage preservation is not being negatively affected by the addition of Bacillus strains in a silage inoculant.

Example 4: On-Farm Application of a Silage Inoculant Containing Bacillus Strains as a Biological Control Agent for Clostridia in Silage

Introduction:

Clostridia fermentation in silage is controlled using lactic acid bacteria as silage inoculants to support the preservation of the crop and by ensuring the crop is harvested and ensiled under low-moisture conditions. However, managing the on-farm conditions such as weather that would impact the moisture content of the crop at harvest is not always practical or possible, and often ensiling occurs under sub-optimal conditions. Although bacilli are considered silage spoilage organisms, members of the Bacillus genera are known to produce antimicrobial compounds capable of inhibiting competing bacteria in the surrounding environment, and have demonstrated efficacy in controlling the growth of clostridia. The purpose of this study was to determine the efficacy of Bacillus strains, in accordance with this embodiment of the present invention, at controlling the growth of clostridia spoilage organisms in alfalfa ensiled on farm and in mini-silos.

Materials and Methods:

The use of a silage inoculant containing Bacillus strains to control clostridia spoilage organisms in preserved forage was investigated to document the efficacy of the inoculant when applied on-farm. Second cut alfalfa haylage was used in the study, and treatments consisted of an untreated control and the silage inoculant applied to the forage on the farm at cutting. The silage inoculant contained 12.5% Bacillus 1104 and 12.5% Bacillus 1781, 7% Enterococcus faecium, 22.5% each of LP 115 (Lactobacillus plantarum), PJ 300 (Pediococcus acidilactici), and P 751 (P. pentosaceus). The target application rate of the silage inoculant was 200,000 CFU/gram consisting of 150,000 CFU lactic acid bacteria (LAB) and 50,000 CFU Bacillus. Each treatment was applied to 1,000 g of forage material and packed in 8 oz glass ball jars with sealed lids at a density of 39 lb/ft³. Jars were stored at room temperature and enumerations were performed on days 0, 7, 14, 28, 58, 91, and 175 days for bacteria of interest (Bacillus, LAB, E. coli and coliforms, clostridia, yeast and mold). Both treated and untreated samples were tested on Day 0 for pH and initial background bacteria present in the samples. At each time point, pH readings were also recorded for each sample and analyzed for presence and concentration of VFAs, including lactic acid, acetic acid, butyric acid, isobutyric acid, propionic acid, valeric acid, and isovaleric acid on an as-sampled basis. VFA results were reported as the average of duplicate values.

Results and Discussion:

During the ensiling process, the pH of the forage samples from both treatments decreased from Day 0 to Day 14 (FIG. 22). This lowered pH level was maintained over the course of the experimental period. This data suggests that the addition of Bacillus in applied silage inoculants can help lower the pH rapidly, starting the fermentation process sooner, and can help maintain a more stable pH during the stable phase (or storage phase) of the ensiling process. A lower pH can help inhibition of spoilage bacteria, improve the digestibility of the feed, reduces proteolytic enzymes, inhibits anaerobic bacteria, shifts fermentation end products, and increases hydrolysis of hemicellulose.

E. coli and coliform populations were reduced from Day 0 to Day 14 in samples from both treatments (FIG. 23; FIG. 24). Following Day 14, E. coli and coliform counts remained at non-detectable limits for the remaining measurements. Both E. coli and coliform levels were lower in the sample administered the bacterial treatment on Day 7 compared to the untreated control sample, indicating that bacterial treatment more effectively controlled these spoilage organisms than the untreated sample.

Bacillus organisms were present as background counts for both treatments at the initial measurement at Day 0 (FIG. 25). By the end of the trial, Bacillus counts were higher when the forage was treated with the bacterial silage inoculant compared to Day 0. Furthermore, the administration of Bacillus represented in the bacterial inoculant did not result in substantially different Bacillus counts over the course of the experimental period compared to the untreated control, indicating the Bacillus administered in the inoculant did not overgrow and act as a spoilage organism. LAB counts were similar between the untreated control and when a bacterial silage inoculant was administered (FIG. 26).

Yeast and mold counts decreased by approximately 5 logs within the first 7 days post ensiling, and remained low (<1.0 E+02 CFU/g) for both treatments throughout the remainder of the experimental period (FIGS. 27 and 28), indicating an effective fermentation process to preserve the forage.

Clostridia counts for the samples treated with inoculant were similar or lower than those of the Control for the entire trial (FIG. 29). This data indicates that the addition of silage inoculants to forage is controlling clostridia growth and secondary fermentation more effectively than when no treatment is administered.

Lactic acid and acetic acid levels were similar between the two treatments throughout the course of the study (FIG. 30; FIG. 31). Some fluctuation was observed in levels of these VFAs on Day 90, but similar values were again observed between the two treatments on the last sampling time point.

Conclusion:

The pH decreased for all treatments in the initial measurement following treatment administration and was maintained over the course of the study. E. coli and coliform counts were reduced in inoculant treatment samples at a faster rate compared to the Control. Treated samples had slightly lower levels of mold present compared to their untreated controls. Clostridia counts for the samples treated with inoculant were similar or lower than those of the Control for the entire trial. This data indicates that the addition of silage inoculants to forage is controlling clostridia growth and secondary fermentation more effectively than when no treatment is administered.

This data indicates that silage preservation is not negatively affected by the addition of these Bacillus strains to a silage inoculant. In fact, the Bacillus in combination with LAB improved the reduction of coliforms and prevented mold growth, more effectively than when no treatment was administered. Clostridial growth and secondary fermentation was controlled more effectively than when no treatment is administered.

Example 5: Selection of Bacillus Strains to Inhibit Clostridium Perfringens and Non-Toxigenic Clostridia Isolated from Ruminant Fecal Samples. (Wisconsin)

Introduction:

Clostridium is a genus of Gram-positive, spore-forming bacteria that are common residents of the gastrointestinal tract. A number of Clostridium species have been linked to enteric disease in ruminants including hemorrhagic bowel syndrome (HBS), a disease often correlated to elevated levels of C. perfringens Type A (Dennison et al., 2005). While most of the enteric diseases caused by clostridia are acute and occur sporadically in herds, in general, the prognosis is poor and the first sign of illness may be death. Based on recent results sub-acute enteric clostridia disease challenges may be a more wide spread issue than acute challenges. Due to a low success rate from treatment in acute disease challenges a more common, emphasis needs to be placed on prophylactic measures.

The purpose of this research was to characterize the distribution and diversity of clostridia in ruminants and ensure inhibition of these isolates using novel Bacillus strains as a method to control the clostridia populations.

Materials and Methods:

Fecal samples (228) from cows, heifers and calves gathered from 24 farms in Wisconsin were diluted 1:10 with sterile peptone, heat shocked for 30 minutes at 60° C., enumerated in sterile peptone and pour plated on Tryptose Sulphite Cycloserine (TSC) agar with D-cycloserine (400 mg/L) to select for clostridia species. Agar plates were incubated at 37° C. anaerobically for 24 hours. If present, isolated sulphite-reducing colonies were counted for a total clostridia count (CFU/g) and representative isolates were picked into Reinforced clostridia Medium (RCM) (Oxoid, CM0149) and incubated anaerobically for 24 hours at 37° C. After 24 hours of incubation the cultures were transferred (10%) to Brain Heart Infusion (BHI) broth (BD, 211059) and incubated anaerobically for 24 hours at 37° C.

DNA extractions were performed in 96-well blocks containing 500 μl presumptive clostridia culture per well. Cells were harvested by centrifugation at 4,700 rpm for 10 minutes, the supernatant was removed. Cells were re-suspended in 500 μl of 50 mM of EDTA-2Na (pH=8.0). Aliquots of 300 μl of the suspended cells were transferred to a new 96-well block and combined with 20 μl of lysozyme from chicken egg white (Sigma, L6876) solution (100 mg/ml in 50 mM EDTA) to lyse bacterial cells. The 96-well block was incubated for 1 hour at 37° C. to lyse bacterial cells. Following the incubation 220 μl of lysis buffer (6 M Guanidine, 20% Triton-X 100, 10 mM Tris-HCL, pH 7.5) was added, mixed then incubated at room temperature for 15 minutes. Following the incubation 20 μl of Proteinase K (NEB, 800 U/mL) was added to each well, mixed and incubated at 55° C. for 30 minutes to degrade proteins. The cell lysate was then transferred to 96-well binding plate (Promega, A2278) and centrifuged at 4,700 rpms for 5 minutes. Flow through was discarded, three washes of the binding plate columns were executed centrifuging 750 μl of Column Wash Solution (Promega, A1318) at 4700 rpms for 1 minute and 30 seconds discarding flow through at the end of each spin. The binding plate was centrifuged for an additional 10 minutes at 4,700 rpm to remove any residual ethanol. A clean elution plate was then placed under the binding plate and DNA was eluted with 200 μl, pre-warmed (55° C.), Nuclease Free Water (Promega, P1195).

DNA was screened for toxin genes (a, 0, c, and t) specific to C. perfringens using polymerase chain reaction (PCR). Amplification of toxin genes was executed using a multiplex PCR containing four primer sets (Yoo et al., 1997) (Table 1.) The PCR mixture contained 2.5 μl 10×PCR Buffer, 2 μl of 50 mM MgCl₂, 0.5 μM of each primer (Table 1.), 0.1 μl of Invitrogen™ Platinum™ Taq DNA Polymerase, 2.5 μl of DNA, sterile water was added to achieve 25 μl for a total reaction volume. The mixture underwent 5 minutes at 94° C., followed by 30 cycles of 94° C. for 1 minute, 55° C. for 1 minute, 72° C. for 1 minute finishing with a final elongation of 3 minutes at 72° C. PCR products were observed using a Fragment Analyzer (Advanced Analytics) to determine if amplification was achieved. If one or multiple toxin genes were observed a toxin type identification was assigned to each isolate based on their toxin gene profile (Songer, 1996). C. perfringens positive to total clostridia isolate ratio was used to calculate an estimated C. perfringens count based on the total clostridia count.

Unique strain-specific genetic fingerprints were generated using Random Amplification of Polymorphic DNA (RAPD) analysis on select isolates to determine diversity among fecal C. perfringens isolates. The PCR contained 5 μl of DNA, 2.5 μl RAPD primer 2 (10 μM) (Table 1.), and 17.5 μl of sterile water which was added to a Ready-To-Go RAPD Analysis Bead (Life Sciences, 27-9500-01). The mixture underwent 5 minutes at 95° C., followed by 45 cycles of 95° C. for 1 minute, 36° C. for 1 minute, 72° C. for 2 minutes finishing with a final elongation of 5 minutes at 72° C. PCR products observed on a Fragment Analyzer (Advanced Analytics) to determine amplification patterns and were imported into BioNumerics, bioinformatics software, for analysis. RAPD patterns were compared with a band based Dice correlation analysis method to determine the similarity between RAPD patterns as a way to monitor diversity between isolates.

Antimicrobial screening was done on C. perfringens isolates obtained from ruminant samples to gauge the effectiveness of the antimicrobial bacteriocin produced by the inventors' identified Bacillus strains 747, 1104, 1541, 1781, and 2018. Bacteriocin was harvested by growing each strain at 32° C. in a shaking incubator at 150 rpms for 24 hours in Brain Heart Infusion (BHI) broth. A 1% transfer of the 24-hour culture to fresh BHI broth was executed after incubation. The Bacillus were then incubated for 36-48 hours in a 32° C. shaking incubator at 150 rpms. The culture was then centrifuged at 14,000×g for 20 minutes, supernatant was then filtered with a 0.2 m filter to remove any residual cells.

A bacteriocin turbidity assay was executed by growing C. perfringens strains isolated from ruminant fecal samples in RCM for 24 hours, anaerobically, at 37° C. Overnight culture was transferred (1%) to sterile RCM and immediately used in the assay. For each C. perfringens isolate at least six wells were run in a sterile 48 well reaction plate, 600 μl inoculated culture (positive control), 600 μl inoculated RCM+70 μl bacteriocin (747, 1104, 1541, 1781, and 2018) and 670 RCM (un-inoculated, negative control). Plates were incubated anaerobically at 37° C. for 24 hours then read using a BioTek Epoch Microplate Spectrophotometer, readings were taken at a wavelength of 600 nm. Optical density readings from the negative controls were subtracted from all OD readings and percent inhibition was calculated using the positive control and each bacteriocin treatment.

To identify clostridia that did not have at least one toxin gene specific to C. perfringens, a PCR reaction was performed on the isolate DNA to amplify the 16S region of rDNA using primers 27F-YM and 1492R-Y (Table 1). This was done on 20% of the isolates that did not contain a toxin gene specific to C. perfringens. The PCR mixture contained 5 μl of 10×PCR Buffer, 2 μl of 50 mM MgCl₂, 1 μl of 50 mM dNTPs, 0.4 μM of each primer (Table 1.), 0.2 μl of Invitrogen™ Platinum™ Taq DNA Polymerase, 5 μl of DNA, and sterile water was added to achieve 50 μl for a total reaction volume. The mixture underwent 4 minutes at 95° C., followed by 35 cycles of 95° C. for 30 seconds, 50° C. for 30 seconds, 72° C. for 2 minutes finishing with a final elongation of 7 minutes at 72° C. A quality check was done on the amplification and PCR product was sent to gene wiz (https://www.genewiz.com) to obtain the sequences for the 16S genes. Sequences were compared to known typed bacterial strains obtained from EZbiocloud online electronic database (http://www.ezbiocloud.net/). Based on comparisons of these sequences a bacterial identification was assigned to the isolates.

A bacteriocin turbidity assay was executed by growing non-toxigenic, non-C. perfringens, clostridia strains isolated from ruminant fecal and feed samples in RCM for 24 hours, anaerobically, at 37° C. Overnight culture was transferred (1%) to sterile RCM and immediately used in the assay. For each C. perfringens isolate at least six wells were run in a sterile 48 well reaction plate, 600 μl inoculated culture (positive control), 600 μl inoculated RCM+70 μl bacteriocin (747, 1104, 1541, 1781, 1999 and 2018) and 670 RCM (un-inoculated, negative control). Plates were incubated anaerobically at 37° C. for 24 hours then read using a BioTek Epoch Microplate Spectrophotometer, readings were taken at a wavelength of 600 nm. Optical density readings from the negative controls were subtracted from all OD readings and percent inhibition was calculated using the positive control and each bacteriocin treatment.

Results:

Fecal samples, 228, were collect from 24 Wisconsin dairy farms from which 2914 presumptive clostridia strains were isolated as representatives of the clostridial diversity in Wisconsin dairies (Table 7.).

Clostridia enumeration results indicated the average level of clostridia CFU/g across all calf fecal samples was 1,240,000 CFU/g with individual fecal samples ranging from <10 to 28,900,000 CFU/g. While the average level of clostridia CFU/g across all cow fecal samples was 59,200 CFU/g with individual fecal samples ranging from 10 to 6,700,000 CFU/g (FIG. 32.). Samples appeared to range in clostridia levels for both calves (FIG. 33.) and cows (FIG. 34.).

C. perfringens enumeration results displayed the average level of C. perfringens CFU/g across all calf fecal samples was 495,000 CFU/g with individual samples ranging from <10 to 14,500,000 CFU/g. While the average level of C. perfringens CFU/g across all cow fecal samples was 36,200 CFU/g with individual samples ranging from <10 to 3,690,000 CFU/g (FIG. 35.). Samples appeared to range in clostridia levels for both calves (FIG. 36.) and cows (FIG. 37.).

Analysis of the toxin multiplex PCR results displayed which isolates contained toxin genes specific to C. perfringens. A total of 1,737 presumptive clostridia isolates from cow fecal samples have been tested for the indicated C. perfringens toxin genes. Of the 1,737 cow clostridia isolates screened, 1,219 isolates (70.2%) tested positive for at least 1 of the toxin genes. From the 1,219 toxin-gene positive isolates 1,203 (98.5%) were identified as Type A (a toxin only), however β, ε and ι, toxins were also detected in the clostridia cow fecal isolates. A total of 1,177 presumptive clostridia isolates from calf fecal samples have been tested for the indicated C. perfringens toxin genes. Of the 1,177 calf clostridia isolates screened 482 (41.0%) tested positive for at least 1 of the toxin genes. From the 482 toxin gene positive isolates 438 (90.9%) were identified as Type A (αtoxin only), however β, ε and ι, toxins were also detected in the clostridia calf fecal isolates.

Gentic RAPD fingerprint patterns displayed diversity among the 1,522 isolates that successful amplified. The isolates tested were harvested from calf fecal, cow fecal and feed and did not cluster strictly based on the sample type or farm. Isolates formed 170 clusters based on 75% similarity according to the Dice correlation method. The largest cluster was 148 isolates (9.7%) and comprised of isolates from 11 different farms, all but two isolates were C. perfringens Type A (FIG. 38.).

Representatives from the RAPD dendrogram were selected to capture the diversity of the C. perfringens population from this region and subjected to inhibition assays. Antimicrobial testing using the bacteriocin turbidity assay displayed good inhibition of most ruminant fecal C. perfringens isolates using bacteriocin harvested from 747, 1104, 1541, 1781, and 2018. The bacteriocin from at least one of the strains 747, 1104, 1541, 1781, and 2018 were able to inhibit the growth >60% of 244 of the 271 isolates tested representing a total of 93.4% inhibition of the C. perfringens population based on the dendrogram (Table 8).

Out of the 2,914 isolates collected 1,207 isolates (41%) were found to be non-toxigenic clostridia. Sequencing representatives (n=183) from the non-toxigenic clostridia displayed three dominate clostridia groups (FIG. 39.). Clostridium bifermentans group (Paraclostridium bifermentans and P. benzoelyticum), Clostridium butyricum and Clostridium beijerinckii group (C. diolis, C. beijerinckii, C. chromiireducens, C. saccharoperbutylacetonicum, C. puniceum, and C. saccharobutylicum), the three main groups of the non-toxigenic clostridia group made up 47.5% of the non-toxigenic isolates (FIG. 40.).

Six isolates with known 16S identifications from Wisconsin were selected for antimicrobial testing using the bacteriocin turbidity assay. Results displayed good inhibition of most ruminant non-toxigenic isolates tested using bacteriocin harvested from 747, 1104, 1541, 1781, 1999 and 2018. The bacteriocin from at least one of the strains 747, 1104, 1541, 1781, 1999 and 2018 were able to inhibit the growth >60% of 6 of the 6 isolates tested (Table 9).

Discussion:

Fecal samples were used as the most readily available sample type to estimate the level and obtain isolates of clostridia and C. perfringens within the digestive system of ruminants. From the 228 fecal samples collected throughout Wisconsin all but two contained detectable levels of clostridia. The survey results indicated clostridia is present in almost all fecal samples at various levels. The majority of the isolates harvested (59%) from the ruminant samples contained a toxin gene specific to C. perfringens. C. perfringens was detected in 92% of the cow fecal samples and in 77% of the calf fecal samples. The high presence of clostridia and C. perfringens indicates the risk for sub-acute enteric clostridia disease challenges in most ruminants throughout Wisconsin. C. perfringens isolates were diverse according to the RAPD genetic fingerprints but were not specific to sample type or farm. Diverse representatives of C. perfringens were mostly inhibited (>60%) by at least one bacteriocin from the following strains 747, 1104, 1541, 1781 or 2018. From the 271 isolates tested 244 isolates were inhibited by greater than 60% by at least one of the strains, representing inhibition of 93.4% of the total C. perfringens population based on representation from clusters on the RAPD dendrogram. This indicates the Bacillus strains 747, 1104, 1541, 1781 and 2018 can inhibit a wide range of diversity of C. perfringens isolates. The Bacillus strains are not limited to specific clostridia strain(s) like a vaccine which may be missing large groups of the clostridia populations based on the genetic diversity observed in the RAPD dendrogram. DNA sequencing of the non-toxigenic clostridia revealed three major identifications of Clostridium species. C. bifermentans group, which is known to produce 1,3-propanediol (Leja et al., 2014; Myszka et al., 2012) C. beijerinckii group known to produce butanol and acetone (Hou et al., 2017), and C. butyricum (Weng et al., 2015) producer of butyrate. The production of the metabolic end products of these species could be having an impact in the rumen, reducing performance parameters such as milk production within a dairy cow. Non-toxigenic clostridia tested were all inhibited (>60%) by the bacteriocin from at least one of the following strains 747, 1104, 1541, 1781, 1999 or 2018. The inhibition of the non-toxigenic clostridia could also be a potential mode of action that Bacillus strains are using to increase performance parameters. The high inhibition level against the clostridia isolates in vitro indicates a potential mode of action of the Bacillus strains 747, 1104, 1541, 1781, 1999 and 2018.

The Bacillus strains offer a prophylactic effect on the clostridia populations which may not only increase rumen efficiency leading to increased milk production, but prevent acute levels of C. perfringens reducing the occurrence of digestive deaths. The high prevalence of clostridia and C. perfringens in fecal samples collected suggests efficiency improvement opportunities in many ruminants throughout Wisconsin. This example displays the diversity of clostridia isolates from the ruminant fecal and feed samples collected from Wisconsin. The Bacillus strains tested 747, 1104, 1541, 1781, 1999 and 2018, could inhibit most of the clostridia diversity observed in Wisconsin. The product, in accordance with this embodiment of the present invention could inhibit both toxigenic and non-toxigenic clostridia isolated from ruminants in Wisconsin indicating a benefit in rumen efficiency if fed to dairy cows as a direct fed microbial (DFM).

TABLE 7 Fecal samples, 228, were collected from Wisconsin dairies separated by farm (24) and age (cow or calf) which were enumerated for clostridia, tested for C. perfringens, isolates were genotyped and tested for inhibition. Farm Name Cow Fecal Calf Fecal Farm WB 54 4 Farm CR 6 4 Farm G 8 2 Farm S 4 4 Farm DE 8 2 Farm ME 3 6 Farm MC 3 8 Farm BE 6 2 Farm GR 4 4 Farm LP 4 4 Farm AL 3 4 Farm Z 4 5 Farm HN 4 4 Farm MV 4 4 Farm HA 3 0 Farm WO 0 7 Farm AD 2 2 Farm MI 4 4 Farm BA 4 4 Farm R 0 1 Farm OO 0 9 Farm GO 0 8 Farm CW 5 0 Farm CH 0 3 Total 133 95

TABLE 8 Bacteriocin assay results displaying each isolate tested and the source of that isolate, isolated from Wisconsin fecal samples. Inhibition was calculated based on the percent of growth for each treatment well compared to the positive control. Isolate Sample Bacillus Strains ID Farm Type Group 747 1104 1541 1781 2018 PA65-2 Farm Calf Calf   98%   98%   98%   98%   99% AD Fecal PA65-3 Farm Calf Calf   100%   71%   98%   98%   100% AD Fecal PA66-1 Farm Calf Calf   86%   100%   100%   100%   100% AD Fecal PA66- Farm Calf Calf    0%    0%    0%    0%    0% 16 AD Fecal PA66-8 Farm Calf Calf   100%   100%   100%   100%   100% AD Fecal PA68-1 Farm Cow Mature Cow   100%   100%   100%   100%   100% AD Fecal PA66- Farm Calf Calf  99.7%  99.9%  99.8%  99.8%  99.8% 11 AD Fecal PA67- Farm Cow Prefresh  83.9%  80.3%  88.8%  89.5%  88.0% 12 AD Fecal PA68- Farm Cow Mature  49.5%  48.2%  51.5%  51.9%  51.2% 16 AD Fecal PA68-2 Farm Cow Mature  99.9%  99.2%  99.8%  99.1%  99.8% AD Fecal PA3-6 Farm Calf Calf   100%   100%   100%   100%   100% AL Fecal PA3-7 Farm Calf Calf   100%   99%   100%   100%   100% AL Fecal PA4-5 Farm Calf Calf    2%    9%    4%    9%    8% AL Fecal PA5-8 Farm Calf Calf   100%   94%   99%   99%   100% AL Fecal PA7-11 Farm Cow Cow Fecal   100%   92%   94%   98%   95% AL Fecal PA7-15 Farm Cow Cow Fecal   61%   43%   41%   69%   59% AL Fecal PA7-2 Farm Cow Cow Fecal   32%    0%    0%   32%    6% AL Fecal PA7-5 Farm Cow Cow Fecal   100%   97%   98%   100%   98% AL Fecal PA8-1 Farm Cow Cow Fecal   98%   90%   93%   97%   97% AL Fecal PA3-2 Farm Calf Calf  98.6%  72.4%  85.1%  75.8%  95.1% AL Fecal PA3-5 Farm Calf Calf  99.8%  99.8%  99.8%  99.9%  99.8% AL Fecal PA4-1 Farm Calf Calf  81.3%  82.0%  82.0%  81.7%  82.0% AL Fecal PA4-3 Farm Calf Calf  97.7%  97.3%  97.5%  97.5%  97.9% AL Fecal PA5-12 Farm Calf Calf  99.4%  95.7%  99.7%  99.6%  99.1% AL Fecal PA5-3 Farm Calf Calf  99.8%  99.9%  99.9%  99.6%  99.9% AL Fecal PA5-4 Farm Calf Calf  0.0%  25.9%  0.0%  53.1%  45.9% AL Fecal PA5-5 Farm Calf Calf  99.2%  99.6%  99.5%  99.6%  99.4% AL Fecal PA5-6 Farm Calf Calf  89.4%  49.8%  48.8%  70.0%  71.7% AL Fecal PA5-7 Farm Calf Calf  85.6%  50.5%  36.8%  65.9%  45.1% AL Fecal PA5-9 Farm Calf Calf  99.8%  85.4%  92.9%  97.3%  99.6% AL Fecal PA6-2 Farm Cow Cow Fecal  98.9%  85.2%  88.0%  94.2%  88.6% AL Fecal PA42- Farm Calf Calf   94%   54%   92%   88%   97% 19 BA Fecal PA46-8 Farm Cow 1st Lactation   22%   25%   25%   24%   25% BA Fecal PA47- Farm Cow 3rd Lactation   24%   25%   26%   25%   26% 10 BA Fecal PA48-4 Farm Cow Mature   100%   100%   100%   100%   100% BA Fecal PA41- Farm Calf Calf 100.0%  9.1% 100.0%  79.8%  99.9% 11 BA Fecal PA42- Farm Calf Calf 100.0%  52.7%  70.6%  87.2%  82.0% 11 BA Fecal PA9-9 Farm Calf Calf  19.8%  23.4%  16.6%  20.5%  19.7% BE Fecal PA11-1 Farm Cow Heifer   100%   96%   100%   100%   100% BE Fecal PA11- Farm Cow Heifer   75%   58%   55%   85%   73% 12 BE Fecal PA11-2 Farm Cow Heifer   100%   100%   100%   100%   100% BE Fecal PA14- Farm Cow Fresh   86%   84%   85%   97%   82% 14 BE Fecal PA14- Farm Cow Fresh   98%   96%   97%   97%   97% 15 BE Fecal PA10-5 Farm Calf Calf  7.7%  6.2%  2.6%  12.4%  8.7% BE Fecal PA11- Farm Cow Heifer  99.9%  99.8% 99.1%  99.9% 100.0% 13 BE Fecal PA11- Farm Cow Heifer  99.5%  99.8%  99.7%  99.8%  99.7% 19 BE Fecal PA11-5 Farm Cow Heifer  99.2%  93.4%  95.1%  96.9%  92.0% BE Fecal PA11-6 Farm Cow Heifer  99.7% 100.0%  99.7%  99.7%  99.7% BE Fecal PA11-7 Farm Cow Heifer 100.0% 100.0% 100.0% 100.0% 100.0% BE Fecal PA12-1 Farm Cow Heifer 100.0%  33.4%  62.2%  70.3%  94.9% BE Fecal PA13- Farm Cow Fresh  38.4%  36.8%  40.9%  40.6%  39.3% 10 BE Fecal PA13- Farm Cow Fresh  99.6%  48.9%  66.6%  45.7%  77.9% 12 BE Fecal PA13- Farm Cow Fresh  99.7% 100.0%  99.9%  99.8%  99.8% 15 BE Fecal PA13- Farm Cow Fresh  99.7%  95.5%  98.2%  99.3%  99.9% 19 BE Fecal PA13-9 Farm Cow Fresh  99.9%  99.9%  99.9%  99.9%  99.8% BE Fecal PA14-1 Farm Cow Fresh  48.1%  0.0%  1.6%  4.7%  4.7% BE Fecal PA14- Farm Cow Fresh  99.9%  99.8%  99.8%  99.9%  99.9% 18 BE Fecal PA14-2 Farm Cow Fresh  99.7%  99.6%  99.7%  99.6%  99.8% BE Fecal PA14-3 Farm Cow Fresh  99.7%  99.8%  99.8%  99.8%  99.8% BE Fecal PA14-4 Farm Cow Fresh 100.0% 100.0% 100.0% 100.0% 100.0% BE Fecal PA15- Farm Cow 2nd year Cow  99.6%  84.3%  79.1%  87.2%  86.3% 17 BE Fecal PA16-8 Farm Cow Mature  0.0%  0.0%  0.0%  0.0%  0.0% BE Fecal 30.4.2 Farm Feed Corn Silage   43%   43%   46%   45%   43% CW B7 PA85-3 Farm Cow Fresh   100%   100%   100%   100%   100% CW Fecal PA87-1 Farm Cow Fresh    0%    0%    0%    0%    0% CW Fecal PA50-2 Farm Calf Calf   100%   100%   100%   100%   100% GR Fecal 21.2.3 Farm Feed Haylage B3   100%   98%   74%   99%   97% GR 21.2.5 Farm Feed Haylage B3   98%   49%   42%   94%   48% GR 21.2.7 Farm Feed Haylage B3   98%   96%   96%   98%   96% GR 21.2.4 Farm Feed Haylage  33.9%  5.2%  4.7%  2.2%  5.3% GR PA55- Farm Cow Fresh  99.7% 100.0%  99.8%  99.7%  99.8% 14 GR Fecal PA55- Farm Cow Fresh  99.8%  99.9%  99.7%  99.8%  99.8% 19 GR Fecal PA55-6 Farm Cow Fresh  99.8%  99.9%  99.9%  99.9%  99.9% GR Fecal PA56- Farm Cow Fresh 99.3%  99.7%  99.7%  99.7%  99.5% 20 GR Fecal J2-12 Farm Cow Lactating  99.9% 100.0% 100.0%  99.9% 100.0% HA Fecal CSS-4 Farm Feed Corn Silage  29.5%  31.7%  96.2%  38.3%  28.6% HN HSS-1 Farm Feed Haylage  99.9% 100.0%  99.9% 100.0%  99.9% HN 28.1.2 Farm Feed Ryelage   64%   63%   91%   63%   69% HN PA69-1 Farm Calf Calf   95%   91%   53%   100%   99% HN Fecal PA69-2 Farm Calf Calf   99%   16%   64%   92%   98% HN Fecal PA69- Farm Calf Calf  99.9%  99.8%  99.9%  99.9% 100.0% 12 HN Fecal PA71- Farm Calf Calf 97.1%  97.8%  96.5%  97.2%  97.5% 12 HN Fecal PA74-1 Farm Cow Fresh 100.0% 100.0% 100.0% 100.0% 100.0% HN Fecal PA74- Farm Cow Fresh 100.0%  0.0%  0.0%  99.7%  45.4% 11 HN Fecal PA74- Farm Cow Fresh 100.0% 100.0% 100.0% 100.0% 100.0% 12 HN Fecal PA74- Farm Cow Fresh 100.0%  26.3%  40.0%  29.0%  72.4% 15 HN Fecal PA74-4 Farm Cow Fresh 100.0%  49.4%  18.7%  73.1%  98.6% HN Fecal PA76-4 Farm Cow Lactating  98.3%  99.1%  98.8%  99.1%  99.0% HN Fecal PA76- Farm Cow Lactating   100%   100%   100%   100%   100% 11 HN Fecal PA76- Farm Cow Lactating   100%   100%   100%   100%   100% 17 HN Fecal PA76- Farm Cow Lactating   100%   100%   100%   100%   100% 19 HN Fecal A1-17 Farm Calf Calf 98.2%  74.1%  47.4%  95.7%  62.3% GO Fecal A1-11 Farm Calf Calf  99.6% 100.0%  99.7%  99.7% 100.0% GO Fecal A1-16 Farm Calf Calf  97.2%  62.1%  51.2%  94.5%  49.9% GO Fecal A2-12 Farm Calf Calf  96.3%  26.2%  59.9%  98.2%  23.3% GO Fecal A2-4 Farm Calf Calf  86.7%  47.4%  68.3%  63.3%  72.3% GO Fecal A2-5 Farm Calf Calf  97.6%  66.3%  65.7%  96.2%  70.0% GO Fecal A2-7 Farm Calf Calf  79.0%  13.4%  24.2%  39.2%  43.0% GO Fecal A2-8 Farm Calf Calf  99.4%  95.4%  94.9%  98.2%  93.6% GO Fecal A7-1 Farm Calf Calf  99.8%  99.9% 100.0%  99.6% 100.0% GO Fecal A7-11 Farm Calf Calf  90.9%  32.7%  16.8%  59.6%  28.6% GO Fecal A7-17 Farm Calf Calf  99.9%  99.9% 100.0%  99.9%  99.9% GO Fecal PA35-4 Farm Calf Calf   100%   100%   100%   100%   100% LP Fecal PA37-8 Farm Cow Lactating   100%   100%   100%   100%   100% LP Fecal PA39-8 Farm Cow Lactating   17%   29%   13%   56%   25% LP Fecal PA33-9 Farm Calf Calf  96.6%  59.3%  74.4%  78.0%  81.2% LP Fecal PA35-1 Farm Calf Calf  99.7%  99.9%  99.8%  99.8%  99.7% LP Fecal PA35- Farm Calf Calf  99.5%  99.9%  99.8%  99.7%  99.8% 10 LP Fecal PA35- Farm Calf Calf  99.9%  99.9%  99.8% 100.0%  99.9% 12 LP Fecal PA36- Farm Calf Calf  99.7% 100.0%  99.7%  99.9%  99.8% 13 LP Fecal PA38- Farm Cow Lactating  99.8% 100.0%  99.9% 100.0%  99.9% 17 LP Fecal PA40-2 Farm Cow Lactating  99.6%  99.7%  99.8%  99.6%  99.9% LP Fecal 3.1.3 Farm Feed Corn Silage   100%   100%   100%   100%   100% MI 3.1.4 Farm Feed Corn Silage   72%   68%   91%   70%   80% MI 31.2.1 Farm Feed Haylage   43%   37%   36%   41%   41% MI PA31- Farm Cow Cow Fecal   100%   100%   100%   100%   100% 16 MI Fecal PA32- Farm Cow Cow Fecal   22%   21%   24%   25%   18% 10 MI Fecal PA32- Farm Cow Cow Fecal   100%    8%   100%   89%   91% 11 MI Fecal PA31-2 Farm Cow Cow Fecal  99.8%  88.5%  89.6%  89.4%  94.7% MI Fecal PA31-5 Farm Cow Cow Fecal 100.0% 100.0%  99.9% 100.0% 100.0% MI Fecal PA31-6 Farm Cow Cow Fecal 100.0% 100.0%  99.9% 100.0% 100.0% MI Fecal PA32- Farm Cow Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% 15 MI Fecal PA32-6 Farm Cow Cow Fecal  99.9% 100.0%  99.9%  99.8%  99.9% MI Fecal 27.2.11 Farm Feed Haylage   98%   91%   81%   95%   91% MV 27.3.2 Farm Feed Oatlage   93%   66%   66%   80%   75% MV 27.3.3 Farm Feed Oatlage   100%   100%   100%   100%   100% MV PA80-5 Farm Calf Calf   100%   100%   100%   100%   100% MV Fecal PA82-2 Farm Cow Fresh   97%   98%   97%   97%   98% MV Fecal PA83-7 Farm Cow Lactating   89%   18%    0%   70%   17% MV Fecal PA84- Farm Cow Lactating   100%   98%   100%   100%   100% 14 MV Fecal PA84- Farm Cow Lactating   100%   100%   97%   100%   100% 18 MV Fecal PA84-7 Farm Cow Lactating   100%   99%   100%   100%   100% MV Fecal PA77-4 Farm Calf Calf  81.7%  77.6%  71.3%  76.9%  82.7% MV Fecal PA80-2 Farm Calf Calf 100.0% 100.0% 100.0% 100.0%  99.7% MV Fecal PA81-3 Farm Cow Fresh  98.4%  98.5%  82.0%  97.8%  98.7% MV Fecal PA81-8 Farm Cow Fresh 100.0%  99.9%  99.9%  99.9%  99.9% MV Fecal PA82- Farm Cow Fresh  99.8%  99.8%  99.7%  99.8%  99.9% 11 MV Fecal PA82-4 Farm Cow Fresh 100.0%  99.9%  99.7%  99.9%  99.9% MV Fecal PA83- Farm Cow Lactating  99.7%  99.9%  99.6%  99.8%  99.7% 16 MV Fecal PA83- Farm Cow Lactating  99.9%  99.8%  99.7%  99.9%  99.7% 19 MV Fecal PA83-2 Farm Cow Lactating  99.6%  99.8%  99.8%  99.8%  99.8% MV Fecal PA84-5 Farm Cow Lactating  99.1%  98.6%  98.8%  98.8%  98.7% MV Fecal PA84-6 Farm Cow Lactating  99.7%  99.8%  99.8%  99.8%  99.9% MV Fecal B1-1 Farm Calf Calf  45.2%  46.0%  48.7%  48.1%  51.7% OO Fecal B1-15 Farm Calf Calf  32.7%  0.0%  26.2%  0.0%  27.0% OO Fecal B3-6 Farm Calf Calf  79.3%  48.7%  54.1%  59.5%  59.9% OO Fecal B4-2 Farm Calf Calf 100.0% 100.0% 100.0% 100.0% 100.0% OO Fecal B4-6 Farm Calf Calf  99.9%  99.9% 100.0%  99.9%  99.8% OO Fecal PA1-2 Farm R Calf Calf  99.7%  84.2%  99.8%  99.6% 100.0% Fecal PA1-3 Farm R Calf Calf  99.8% 100.0%  99.8% 100.0% 100.0% Fecal PA1-6 Farm R Calf Calf 100.0%  41.0%  86.0%  86.2% 100.0% Fecal PA1-10 Farm R Calf Calf 100.0%  26.1%  80.4%  76.1% 100.0% Fecal PA1-11 Farm R Calf Calf 100.0%  8.7%  81.1%  78.4% 100.0% Fecal PA1-16 Farm R Calf Calf 100.0%  38.5%  82.4%  77.7% 100.0% Fecal PA1-19 Farm R Calf Calf 100.0%  0.6%  80.3%  65.5% 100.0% Fecal PA1-20 Farm R Calf Calf  99.9%  16.9%  83.2%  76.1% 100.0% Fecal RF-1 Farm R Feed Corn Silage 100.0% 100.0% 100.0% 100.0% 100.0% ROL-1 Farm R Feed Haylage-Old  92.6%  94.7%  94.5%  94.4%  89.5% ROL-3 Farm R Feed Haylage-Old  99.9% 100.0%  99.8%  99.9%  99.7% ROS-1 Farm R Feed Haylage-Old  35.9%  16.1%  17.4%  17.0%  18.5% ROS-3 Farm R Feed Haylage-Old 100.0%  20.3%  28.9%  25.1%  50.5% 20.1.3 Farm S Feed Corn Silage   12%   12%   11%   17%   12% B1 20.1.5 Farm S Feed Corn Silage   21%   18%   37%   25%   19% B1 20.3.1 Farm S Feed Haylage B6   85%   89%   84%   90%   80% 20.4.6 Farm S Feed Corn Silage   100%   100%   100%   100%   100% B7 PA57- Farm S Calf Calf   100%   100%   100%   100%   100% 14 Fecal PA57-7 Farm S Calf Calf   100%   100%   100%   100%   100% Fecal PA58- Farm S Calf Calf   100%   100%   100%   100%   100% 11 Fecal PA59- Farm S Calf Calf   100%   100%   100%   100%   100% 11 Fecal PA63- Farm S Cow Lactating   100%   100%   100%   100%   100% 17 Fecal 20.3.3 Farm S Feed Haylage  90.0%  92.7%  94.1%  93.1%  93.0% 20.3.5 Farm S Feed Haylage  99.7% 100.0%  99.9%  99.9% 100.0% PA57-9 Farm S Calf Calf  99.7% 100.0%  99.9%  99.9% 100.0% Fecal PA58-1 Farm S Calf Calf  99.5% 100.0%  99.8%  99.8%  99.8% Fecal PA58- Farm S Calf Calf  99.9% 100.0% 100.0% 100.0% 100.0% 19 Fecal PA61-3 Farm S Cow Fresh  99.8%  99.9%  99.8%  99.9%  99.8% Fecal PA62- Farm S Cow Fresh  99.7%  99.9%  99.8%  99.9%  99.9% 16 Fecal PA62-5 Farm S Cow Fresh  99.2%  99.5%  99.4%  99.6%  99.4% Fecal PA63- Farm S Cow Lactating 100.0% 100.0% 100.0%  99.9%  99.9% 10 Fecal PA63- Farm S Cow Lactating 100.0%  99.9% 100.0%  99.8% 100.0% 19 Fecal PA23-3 Farm Cow Cow Fecal   100%   100%   100%   100%   100% WB Fecal PA23-5 Farm Cow Cow Fecal   100%   100%   100%   100%   100% WB Fecal PA24-7 Farm Cow Cow Fecal   100%   68%    0%   100%   100% WB Fecal PA21-1 Farm Cow Cow Fecal  99.9% 100.0%  99.9%  99.9%  99.9% WB Fecal PA21- Farm Cow Cow Fecal  99.5%  99.6% 99.6%  99.5%  99.6% 20 WB Fecal PA22-1 Farm Cow Cow Fecal  99.6%  99.7%  99.7%  99.6% 99.6% WB Fecal PA22-3 Farm Cow Cow Fecal  99.4%  99.6%  99.4%  99.6%  99.5% WB Fecal PA23- Farm Cow Cow Fecal  99.7%  99.9%  99.6%  99.9%  99.9% 13 WB Fecal PA23- Farm Cow Cow Fecal  99.8% 100.0% 100.0%  99.9% 100.0% 14 WB Fecal PA23- Farm Cow Cow Fecal  99.8%  99.9%  99.8%  99.8%  99.8% 15 WB Fecal PA24- Farm Cow Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% 19 WB Fecal C1-12 Farm Calf Calf  97.5%  92.7%  90.8%  96.7%  91.2% WO Fecal C1-15 Farm Calf Calf  99.8%  99.9% 100.0%  99.7% 100.0% WO Fecal C1-16 Farm Calf Calf  98.8% 100.0% 100.0% 100.0% 100.0% WO Fecal C1-2 Farm Calf Calf  95.0%  79.0%  79.5%  91.3%  79.6% WO Fecal C1-4 Farm Calf Calf 100.0% 100.0% 100.0% 100.0% 100.0% WO Fecal C1-7 Farm Calf Calf  98.2%  93.3%  92.8%  96.8%  92.3% WO Fecal C1-9 Farm Calf Calf 100.0%  99.9% 100.0%  99.9% 100.0% WO Fecal C2-7 Farm Calf Calf  99.9% 100.0%  99.9%  99.8% 100.0% WO Fecal C2-9 Farm Calf Calf  98.0%  94.2%  93.7%  93.2%  93.4% WO Fecal C3-16 Farm Calf Calf  99.9% 100.0% 100.0%  99.8% 100.0% WO Fecal C3-17 Farm Calf Calf  99.9% 100.0% 100.0% 100.0% 100.0% WO Fecal C3-18 Farm Calf Calf  98.3%  43.0%  95.0%  97.7%  93.5% WO Fecal C3-8 Farm Calf Calf 100.0% 100.0% 100.0% 100.0% 100.0% WO Fecal C4-19 Farm Calf Calf  99.3%  94.7%  94.0%  98.3%  92.8% WO Fecal C5-18 Farm Calf Calf  99.7%  93.1%  87.8%  98.6%  93.0% WO Fecal BT10-9 Farm Cow Dry 98.52% 98.11% 96.23% 95.55% 96.23% WB Fecal BT1-2 Farm Cow Pre-Fresh 75.64% 65.36% 69.74% 69.89% 71.41% WB Fecal BT13-6 Farm Cow Post Fresh 98.44% 95.10% 95.32% 92.20% 93.10% WB Fecal BT16- Farm Cow Late 98.73% 99.37% 98.99% 98.99% 99.11% 10 WB Fecal Lactation BT18-2 Farm Cow Late 86.00% 66.00% 50.00% 26.00% 38.00% WB Fecal Lactation BT18-3 Farm Cow Late 98.25% 94.99% 93.73% 91.73% 92.48% WB Fecal Lactation BT19- Farm Cow Late 98.82% 36.74% 95.48% 93.52% 94.30% 10 WB Fecal Lactation BT20-7 Farm Cow Late 99.47% 99.47% 99.21% 98.95% 99.08% WB Fecal Lactation BT21-8 Farm Cow Post Fresh 99.14% 99.28% 98.99% 98.99% 99.14% WB Fecal BT2-2 Farm Cow Pre-Fresh 89.90% 99.05% 85.13% 97.41% 98.50% WB Fecal BT22-5 Farm Cow Post Fresh 98.77% 98.77% 98.42% 98.25% 98.42% WB Fecal BT22-8 Farm Cow Post Fresh 94.64% 96.43% 93.75% 94.64% 93.75% WB Fecal BT23-5 Farm Cow Post Fresh 99.38% 99.00% 99.00% 99.13% 99.13% WB Fecal BT23-8 Farm Cow Post Fresh 98.77% 98.77% 98.43% 98.65% 98.20% WB Fecal BT24-2 Farm Cow Post Fresh 90.59% 92.94% 89.41% 90.59% 90.59% WB Fecal BT24-3 Farm Cow Post Fresh 96.27% 95.52% 94.78% 94.03% 96.27% WB Fecal BT27-9 Farm Cow Breeding 99.17% 99.31% 99.03% 98.89% 99.03% WB Fecal BT28-4 Farm Cow Breeding 98.79% 99.46% 99.06% 99.06% 99.33% WB Fecal BT28-6 Farm Cow Breeding 98.44% 98.83% 98.64% 98.64% 98.83% WB Fecal BT29-5 Farm Cow Breeding 83.90%  0.00% 21.95%  0.00% 33.17% WB Fecal BT29-7 Farm Cow Breeding 86.35% 78.59% 68.47% 79.76% 58.35% WB Fecal BT31-1 Farm Cow Breeding 99.09% 99.39% 99.24% 99.09% 98.93% WB Fecal BT31-2 Farm Cow Breeding 97.63% 97.89% 97.89% 97.63% 97.89% WB Fecal BT32-8 Farm Cow Breeding 98.33% 18.87% 98.63% 96.50% 96.35% WB Fecal BT35-4 Farm Cow Breeding 87.73% 51.72% 40.10% 57.45% 60.23% WB Fecal BT36-1 Farm Cow Breeding 90.79% 52.63% 6.58% 36.84% 51.32% WB Fecal BT36-3 Farm Cow Breeding 85.67% 62.39% 66.42% 65.67% 73.13% WB Fecal BT36-6 Farm Cow Breeding 98.13% 98.75% 96.88% 96.57% 98.44% WB Fecal BT37-4 Farm Cow Breeding 79.45% 16.60%  0.00% 34.78% 66.80% WB Fecal BT37-5 Farm Cow Breeding 98.67% 28.79% 78.60% 72.54% 98.30% WB Fecal BT3-8 Farm Cow Pre-Fresh 94.70% 94.70% 50.00% 19.70% 86.36% WB Fecal BT3-9 Farm Cow Pre-Fresh 98.46% 83.82% 97.30% 94.99% 94.99% WB Fecal BT39- Farm Cow Breeding 98.00% 98.75% 98.50% 98.50% 98.50% 10 WB Fecal BT39-7 Farm Cow Breeding 98.17% 97.87% 95.73% 96.34% 97.56% WB Fecal BT40-4 Farm Cow Breeding 93.63% 71.04% 97.75% 96.50% 96.13% WB Fecal BT41-1 Farm Cow Breeding/Preg 98.66% 97.05% 98.93% 97.05% 99.20% WB Fecal BT41-2 Farm Cow Breeding/Preg 87.56% 52.86% 60.07% 72.50% 61.05% WB Fecal BT42-7 Farm Cow Breeding/Preg 49.16% 31.14% 52.02% 55.89% 66.16% WB Fecal BT43- Farm Cow Breeding/Preg 98.99% 76.71% 98.99% 98.99% 50.73% 10 WB Fecal BT43-8 Farm Cow Breeding/Preg 98.05% 98.05% 97.40% 98.05% 98.05% WB Fecal BT45-4 Farm Cow Breeding/Preg 99.25% 98.95% 99.10% 99.10% 98.95% WB Fecal BT47-3 Farm Cow Bred  0.00% 0.00%  0.00% 0.00%  0.00% WB Fecal BT48-6 Farm Cow Bred 98.89% 99.45% 99.17% 99.03% 99.31% WB Fecal BT48-8 Farm Cow Bred 99.19% 99.19% 99.19% 99.06% 99.19% WB Fecal BT49-9 Farm Cow Bred 94.41% 41.04% 63.53% 93.39% 93.39% WB Fecal BT50-2 Farm Cow Bred 35.46% 32.57% 24.20% 40.94% 32.27% WB Fecal BT50-3 Farm Cow Bred 99.64% 99.45% 99.55% 99.27% 99.45% WB Fecal BT50-5 Farm Cow Bred 98.59% 99.12% 98.59% 98.59% 99.12% WB Fecal BT6-6 Farm Cow Dry 95.59% 97.94% 95.74% 96.04% 96.92% WB Fecal BT6-7 Farm Cow Dry 98.86% 98.37% 98.53% 97.88% 98.53% WB Fecal BT8-6 Farm Cow Dry  3.66% 45.12% 45.93%  0.00% 96.75% WB Fecal BT9-4 Farm Cow Dry 98.82% 98.97% 98.82% 98.82% 98.97% WB Fecal BT8-3 Farm Cow Dry 35.80% 32.67% 18.75% 48.86% 29.55% WB Fecal BT10-5 Farm Cow Dry 99.89% 99.89% 99.79% 98.72% 99.79% WB Fecal BT21-2 Farm Cow Post Fresh 99.05% 99.29% 98.35% 99.29% 99.05% WB Fecal BT24-9 Farm Cow Post Fresh 98.96% 99.22% 98.69% 98.96% 98.69% WB Fecal BT31-1 Farm Cow Breeding 99.20% 99.52% 99.20% 99.20% 97.92% WB Fecal BT41-7 Farm Cow Breeding/Preg 99.61% 99.61% 99.61% 99.42% 99.42% WB Fecal BT42-4 Farm Cow Breeding/Preg 99.71% 89.80% 99.57% 99.71% 99.43% WB Fecal BT43-5 Farm Cow Breeding/Preg 99.58% 99.79% 99.58% 99.15% 99.36% WB Fecal BT43-9 Farm Cow Breeding/Preg  0.00% 0.00%  0.00% 0.00%  0.00% WB Fecal BT45-7 Farm Cow Breeding/Preg 98.86% 99.71% 98.57% 99.14% 98.86% WB Fecal BT48-2 Farm Cow Bred 99.29% 99.82% 99.47% 99.47% 99.47% WB Fecal BT8-2 Farm Cow Dry 99.45% 98.53% 99.26% 96.51% 99.45% WB Fecal BT9-5 Farm Cow Dry 99.70% 99.55% 99.55% 99.39% 95.61% WB Fecal

TABLE 9 Bacteriocin assay results displaying each isolate tested from Wisconsin. Inhibition was calculated based on the percent of growth for each treatment well compared to the positive control. Bacillus Strains Clostridium sp. 747 1104 1541 1781 1999 2018 C. 95.4% 70.5% 69.6% 69.3% 75.1% 69.6% bifermentans C. 85.1% 46.1% 58.4% 89.6% 43.3% 61.4% bifermentans C. 97.2% 97.4% 97.4% 97.3% 96.0% 98.1% bifermentans C. 72.5% 65.6% 47.0% 68.3% 65.7% 29.1% bifermentans C. 85.9% 48.2% 53.4% 72.4% 46.8% 65.7% bifermentans C. 27.9% 0.3% 14.0% 24.4% 78.2% 24.4% beijerinckii

Example 6: Selection of Bacillus Strains to Inhibit Clostridium Perfringens and Non-Toxigenic Clostridia Isolated from Ruminant Fecal Samples. (Texas)

Introduction:

Clostridium is a genus of Gram-positive, spore-forming bacteria that are common residents of the gastrointestinal tract. A number of Clostridium species have been linked to enteric disease in ruminants including hemorrhagic bowel syndrome (HBS), a disease often correlated to elevated levels of Clostridium perfringens Type A. While most of the enteric diseases caused by clostridia are acute and occur sporadically in herds, in general, the prognosis is poor and the first sign of illness may be death. Based on recent results sub-acute enteric clostridia disease challenges may be a more wide spread issue than acute challenges. Due to a low success rate from treatment in acute disease challenges a more common, emphasis needs to be placed on prophylactic measures.

The purpose of this research was to characterize the distribution and diversity of clostridia in ruminants and ensure inhibition of these isolates using novel Bacillus strains as a method to control the clostridia populations

The objectives were to determine level of clostridia and C. perfringens in cow fecal and feed samples. Determine genotype of non-toxigenic clostridia population. Determine genotype of C. perfringens and test representative isolates sensitivity to the inventors' identified Bacillus strains 747, 1104, 1541, 1781, and 2018. The purpose of this research was to characterize the distribution and diversity of clostridia in ruminants and ensure inhibition of these isolates using novel Bacillus strains as a method to control the clostridia populations.

Materials and Methods:

Fecal samples (827) from cows, heifers and calves gathered from 12 farms in Texas were diluted 1:10 with sterile peptone, heat shocked for 30 minutes at 60° C., enumerated in sterile peptone and pour plated on Tryptose Sulphite Cycloserine (TSC) agar with D-cycloserine (400 mg/L) to select for clostridia species. Agar plates were incubated at 37° C. anaerobically for 24 hours. If present, isolated sulphite-reducing colonies were counted for a total clostridia count (CFU/g) and representative isolates were picked into Reinforced clostridia Medium (RCM) (Oxoid, CM0149) and incubated anaerobically for 24 hours at 37° C. After 24 hours of incubation the cultures were transferred (10%) to Brain Heart Infusion (BHI) broth (BD, 211059) and incubated anaerobically for 24 hours at 37° C.

DNA extractions were performed in 96-well blocks containing 500 μl presumptive clostridia culture per well. Cells were harvested by centrifugation at 4,700 rpm for 10 minutes, the supernatant was removed. Cells were re-suspended in 500 μl of 50 mM of EDTA-2Na (pH=8.0). Aliquots of 300 μl of the suspended cells were transferred to a new 96-well block and combined with 20 μl of lysozyme from chicken egg white (Sigma, L6876) solution (100 mg/ml in 50 mM EDTA) to lyse bacterial cells. The 96-well block was incubated for 1 hour at 37° C. to lyse bacterial cells. Following the incubation 220 μl of lysis buffer (6 M Guanidine, 20% Triton-X 100, 10 mM Tris-HCL, pH 7.5) was added, mixed then incubated at room temperature for 15 minutes. Following the incubation 20 μl of Proteinase K (NEB, 800 U/mL) was added to each well, mixed and incubated at 55° C. for 30 minutes to degrade proteins. The cell lysate was then transferred to 96-well binding plate (Promega, A2278) and centrifuged at 4,700 rpms for 5 minutes. Flow through was discarded, three washes of the binding plate columns were executed centrifuging 750 μl of Column Wash Solution (Promega, A1318) at 4700 rpms for 1 minute and 30 seconds discarding flow through at the end of each spin. The binding plate was centrifuged for an additional 10 minutes at 4,700 rpm to remove any residual ethanol. A clean elution plate was then placed under the binding plate and DNA was eluted with 200 μl, pre-warmed (55° C.), Nuclease Free Water (Promega, P1195).

DNA was screened for toxin genes (α, β, ε, and ι) specific to C. perfringens using polymerase chain reaction (PCR). Amplification of toxin genes was executed using a multiplex PCR containing four primer sets (Yoo et al., 1997) (Table 1.) The PCR mixture contained 2.5 μl 10×PCR Buffer, 2 μl of 50 mM MgCl₂, 0.5 μM of each primer (Table 1.), 0.1 μl of Invitrogen™ Platinum™ Taq DNA Polymerase, 2.5 μl of DNA, sterile water was added to achieve 25 μl for a total reaction volume. The mixture underwent 5 minutes at 940° C., followed by 30 cycles of 94° C. for 1 minute, 55° C. for 1 minute, 72° C. for 1 minute finishing with a final elongation of 3 minutes at 72° C. PCR products were observed using a Fragment Analyzer (Advanced Analytics) to determine if amplification was achieved. If one or multiple toxin genes were observed a toxin type identification was assigned to each isolate based on their toxin gene profile (Songer, 1996). C. perfringens positive to total clostridia isolate ratio was used to calculate an estimated C. perfringens count based on the total clostridia count.

Unique strain-specific genetic fingerprints were generated using Random Amplification of Polymorphic DNA (RAPD) analysis on select isolates to determine diversity among fecal C. perfringens isolates. The PCR contained 5 μl of DNA, 2.5 μl RAPD primer 2 (10 μM) (Table 1.), and 17.5 μl of sterile water which was added to a Ready-To-Go RAPD Analysis Bead (Life Sciences, 27-9500-01). The mixture underwent 5 minutes at 95° C., followed by 45 cycles of 95° C. for 1 minute, 36° C. for 1 minute, 72° C. for 2 minutes finishing with a final elongation of 5 minutes at 72° C. PCR products observed on a Fragment Analyzer (Advanced Analytics) to determine amplification patterns and were imported into BioNumerics, bioinformatics software, for analysis. RAPD patterns were compared with a band based Dice correlation analysis method to determine the similarity between RAPD patterns as a way to monitor diversity between isolates.

Antimicrobial screening was done on C. perfringens isolates obtained from ruminant samples to gauge the effectiveness of the antimicrobial bacteriocin produced by the inventors' identified Bacillus strains 747, 1104, 1541, 1781, and 2018. Bacteriocin was harvested by growing each strain at 32° C. in a shaking incubator at 150 rpms for 24 hours in Brain Heart Infusion (BHI) broth. A 1% transfer of the 24-hour culture to fresh BHI broth was executed after incubation. The Bacillus were then incubated for 36-48 hours in a 32° C. shaking incubator at 150 rpms. The culture was then centrifuged at 14,000×g for 20 minutes, supernatant was then filtered with a 0.2 m filter to remove any residual cells.

A bacteriocin turbidity assay was executed by growing C. perfringens strains isolated from ruminant fecal samples in RCM for 24 hours, anaerobically, at 37° C. Overnight culture was transferred (1%) to sterile RCM and immediately used in the assay. For each C. perfringens isolate at least six wells were run in a sterile 48 well reaction plate, 600 μl inoculated culture (positive control), 600 μl inoculated RCM+70 μl bacteriocin (747, 1104, 1541, 1781, and 2018) and 670 RCM (un-inoculated, negative control). Plates were incubated anaerobically at 37° C. for 24 hours then read using a BioTek Epoch Microplate Spectrophotometer, readings were taken at a wavelength of 600 nm. Optical density readings from the negative controls were subtracted from all OD readings and percent inhibition was calculated using the positive control and each bacteriocin treatment.

To identify clostridia sp. that did not have at least one toxin gene specific to C. perfringens, a PCR reaction was performed on the isolate DNA to amplify the 16S region of rDNA using primers 27F-YM and 1492R-Y (Table 1). This was done on 20% of the isolates that did not contain a toxin gene specific to C. perfringens. The PCR mixture contained 5 μl of 10×PCR Buffer, 2 μl of 50 mM MgCl₂, 1 μl of 50 mM dNTPs, 0.4 μM of each primer (Table 1.), 0.2 μl of Invitrogen™ Platinum™ Taq DNA Polymerase, 5 μl of DNA, and sterile water was added to achieve 50 μl for a total reaction volume. The mixture underwent 4 minutes at 95° C., followed by 35 cycles of 95° C. for 30 seconds, 50° C. for 30 seconds, 72° C. for 2 minutes finishing with a final elongation of 7 minutes at 72° C. A quality check was done on the amplification and PCR product was sent to gene wiz (https://www.genewiz.com) to obtain the sequences for the 16S genes. Sequences were compared to known typed bacterial strains obtained from EZbiocloud online electronic database (http://www.ezbiocloud.net/). Based on comparisons of these sequences a bacterial identification was assigned to the isolates.

A bacteriocin turbidity assay was executed by growing non-toxigenic, clostridia strains isolated from ruminant fecal and feed samples in RCM for 24 hours, anaerobically, at 37° C. Overnight culture was transferred (1%) to sterile RCM and immediately used in the assay. For each C. perfringens isolate at least six wells were run in a sterile 48 well reaction plate, 600 μl inoculated culture (positive control), 600 μl inoculated RCM+70 μl bacteriocin (747, 1104, 1541, 1781, 1999 and 2018) and 670 RCM (un-inoculated, negative control). Plates were incubated anaerobically at 37° C. for 24 hours then read using a BioTek Epoch Microplate Spectrophotometer, readings were taken at a wavelength of 600 nm. Optical density readings from the negative controls were subtracted from all OD readings and percent inhibition was calculated using the positive control and each bacteriocin treatment.

Results:

Fecal samples, 827, were collect from 12 Texas farms from which 7,046 presumptive clostridia strains were isolated as representatives of the clostridial diversity in Texas (Table 10.).

Clostridia enumeration results indicated the average level of clostridia CFU/g across all calf fecal samples was 1,110,000 CFU/g with individual fecal samples ranging from 10 to 17,300,000 CFU/g. While the average level of clostridia CFU/g across all cow fecal samples was 59,700 CFU/g with individual fecal samples ranging from <10 to 35,500,000 CFU/g (FIG. 41.). Samples appeared to range in clostridia levels for both calves (FIG. 42.) and cows (FIG. 43.).

C. perfringens enumeration results displayed the average level of C. perfringens CFU/g across all calf fecal samples was 353,000 CFU/g with individual samples ranging from <10 to 4,020,000 CFU/g. While the average level of C. perfringens CFU/g across all cow fecal samples was 56,100 CFU/g with individual samples ranging from <10 to 35,500,000 CFU/g (FIG. 44.). Samples appeared to range in clostridia levels for both calves (FIG. 45.) and cows (FIG. 46.).

Analysis of the toxin multiplex PCR results displayed which isolates contained toxin genes specific to C. perfringens. A total of 7,046 presumptive clostridia isolates from fecal samples have been tested for the indicated C. perfringens toxin genes. Of the 7,046 clostridia isolates screened, 3,697 isolates (52.5%) tested positive for at least 1 of the toxin genes. From the 3,697 toxin-gene positive isolates 3,588 (97%) were identified as Type A (αtoxin only), however β, ε and ι, toxins were also detected in the clostridia fecal isolates.

Gentic RAPD fingerprint patterns displayed diversity among the 3,460 isolates that successful amplified. The isolates tested were harvested from calf fecal, cow fecal and feed and did not cluster strictly based on the sample type or farm. Isolates formed 513 clusters based on 75% similarity according to the Dice correlation method.

Representatives from the RAPD dendrogram were selected to capture the diversity of the C. perfringens population from this region and subjected to inhibition assays. Antimicrobial testing using the bacteriocin turbidity assay displayed good inhibition of most ruminant fecal C. perfringens isolates using bacteriocin harvested from 747, 1104, 1541, 1781, and 2018. The bacteriocin from at least one of the strains 747, 1104, 1541, 1781, and 2018 were able to inhibit the growth greater than 60% of 877 of the 983 isolates tested representing a total of 92.7% inhibition of the C. perfringens population based on the dendrogram (Table 11.).

Out of the 7,046 isolates collected 3,349 isolates (47.5%) were found to be non-toxigenic clostridia. Sequencing representatives (n=215) from the non-toxigenic clostridia displayed two dominate clostridia groups. Clostridium bifermentans group (Paraclostridium bifermentans and P. benzoelyticum) and Clostridium beijerinckii group (C. diolis, C. beijerinckii, C. chromiireducens, C. saccharoperbutylacetonicum, C. puniceum, and C. saccharobutylicum), the two main groups of the non-toxigenic clostridia group made up 52.1% of the non-toxigenic isolates (FIG. 47.).

Non-toxigenic isolates (n=105) with known 16S identifications from Texas were selected for antimicrobial testing using the bacteriocin turbidity assay. Isolates were selected to cover a diverse representation of non-toxigenic isolates. Results displayed good inhibition of most ruminant non-toxigenic isolates tested using bacteriocin harvested from 747, 1104, 1541, 1781, 1999 and 2018. The bacteriocin from at least one of the strains 747, 1104, 1541, 1781, 1999 and 2018 were able to inhibit the growth >60% for 71 of the 105 isolates tested (Table 12.).

Discussion:

Fecal samples were used as the most readily available sample type to estimate the level and obtain isolates of clostridia and C. perfringens within the digestive system of ruminants. From the 827 fecal samples collected throughout Texas all but one contained a detectable level of clostridia. The survey results indicated clostridia is present in almost all fecal samples at various levels. The majority of the isolates harvested (52.5%) from the ruminant samples contained a toxin gene specific to C. perfringens. C. perfringens was detected in 86% of the cow fecal samples and in 60% of the calf fecal samples. The high presence of clostridia and C. perfringens indicates the risk for sub-acute enteric clostridia disease challenges in most ruminants throughout Texas. C. perfringens isolates were diverse according to the RAPD genetic fingerprints but were not specific to sample type or farm. Diverse representatives of C. perfringens were mostly inhibited (>60%) by at least one bacteriocin from the following strains 747, 1104, 1541, 1781 or 2018. From the 983 isolates tested 877 isolates were inhibited by greater than 60% by at least one of the strains, representing inhibition of 92.7% of the C. perfringens population based on representation from clusters on the RAPD dendrogram. This indicates the Bacillus strains 747, 1104, 1541, 1781 and 2018 can inhibit a wide range of diversity of C. perfringens isolates. The Bacillus strains are not limited to specific clostridia strain(s) like a vaccine which may be missing large groups of the clostridia populations based on the genetic diversity observed in the RAPD dendrogram. DNA sequencing of the non-toxigenic clostridia revealed two major identifications of Clostridium species. C. bifermentans group which is known to produce 1,3-propanediol (Leja et al., 2014; Myszka et al., 2012) and C. beijerinckii group known to produce butanol and acetone (Hou et al., 2017). The production of the metabolic end products of these species could be having an impact in the rumen, reducing performance parameters such as milk production within a dairy cow. Non-toxigenic clostridia isolates tested were mostly inhibited (71/105) greater than 60% by the bacteriocin from at least one of the following strains 747, 1104, 1541, 1781, 1999 or 2018. The inhibition of the non-toxigenic clostridia could also be a potential mode of action that Bacillus strains are using to increase performance parameters. The high inhibition level against the clostridia isolates in vitro indicates a potential mode of action of the Bacillus strains 747, 1104, 1541, 1781, 1999 and 2018.

The Bacillus strains offer a prophylactic effect on the clostridia populations which may not only increase rumen efficiency leading to increased milk production, but prevent acute levels of C. perfringens reducing the occurrence of digestive deaths. The high prevalence of clostridia and C. perfringens in fecal samples collected suggests efficiency improvement opportunities in many ruminants throughout Texas. This example displays the diversity of clostridia isolates from the ruminant fecal and feed samples collected from Texas. The Bacillus strains tested 747, 1104, 1541, 1781, 1999 and 2018, could inhibit most of the clostridia diversity observed in Texas. The product, in accordance with this embodiment of the present invention could inhibit both toxigenic and non-toxigenic clostridia isolated from ruminants in Texas indicating a benefit in rumen efficiency if fed to dairy cows as a direct fed microbial (DFM).

TABLE 10 Fecal samples, 827, were collected from Texas farms separated by farm (12) and age (cow or calf) which were enumerated for clostridia, tested for C. perfringens, isolates were genotyped and tested for inhibition. Farm Name Cow Fecal Calf Fecal Farm DC 0 46 Farm L 70 0 Farm BS 60 0 Farm ALJ 60 0 Farm FC 70 15 Farm RR 70 0 Farm GJJ 70 0 Farm GE 70 0 Farm GH 69 0 Farm DR 82 0 Farm MD 71 15 Farm AX 50 10

TABLE 11 Bacteriocin assay results displaying each isolate tested and the source of that isolate, isolated from Texas fecal samples. Inhibition was calculated based on the percent of growth for each treatment well compared to the positive control. Bacillus strains Isolate ID Sample 747 1104 1541 1781 2018 R2.35.10 Cow Fecal 2.0% 1.5% 0.0% 2.0% 6.1% R2.35.5 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R2.36.2 Cow Fecal 0.0% 0.0% 1.7% 0.0% 0.0% R2.36.3 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R2.36.5 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R2.36.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R2.39.3 Cow Fecal 98.8% 98.6% 98.8% 99.0% 91.3% R2.39.8 Cow Fecal 99.4% 100.0% 100.0% 100.0% 100.0% R2.40.1 Cow Fecal 89.2% 85.7% 87.4% 91.1% 92.0% R2.40.10 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R2.40.6 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R2.40.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R2.42.2 Cow Fecal 98.0% 0.0% 30.1% 0.0% 28.9% R2.42.3 Cow Fecal 0.0% 0.0% 0.0% 0.0% 0.0% R2.42.6 Cow Fecal 11.3% 12.6% 14.0% 15.7% 14.2% R2.44.5 Cow Fecal 14.8% 4.1% 12.9% 13.6% 9.0% R2.45.5 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R2.45.7 Cow Fecal 27.5% 26.6% 28.9% 26.6% 27.5% R2.45.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R2.46.3 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R2.46.5 Cow Fecal 100.0% 100.0% 100.0% 99.8% 100.0% R2.46.6 Cow Fecal 96.8% 68.7% 99.7% 99.7% 99.4% R2.49.5 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R2.50.2 Cow Fecal 1.3% 4.1% 0.0% 0.4% 0.0% R2.50.4 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R2.51.6 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R2.51.7 Cow Fecal 99.9% 100.0% 100.0% 100.0% 100.0% R2.52.2 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R2.52.3 Cow Fecal 100.0% 100.0% 100.0% 100.0% 99.9% R2.52.8 Cow Fecal 99.8% 99.8% 99.9% 99.4% 99.7% R2.54.3 Cow Fecal 36.0% 29.6% 34.2% 35.3% 37.3% R2.56.1 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R2.56.2 Cow Fecal 100.0% 0.0% 0.0% 0.0% 0.0% R2.57.3 Cow Fecal 3.3% 1.5% 3.7% 4.3% 2.7% R2.59.6 Cow Fecal 1.9% 0.0% 0.0% 3.7% 5.4% R2.61.3 Cow Fecal 99.9% 99.2% 100.0% 99.5% 99.9% R2.61.4 Cow Fecal 73.6% 59.0% 51.3% 62.8% 15.9% R2.61.7 Cow Fecal 100.0% 99.6% 100.0% 100.0% 100.0% R2.62.1 Cow Fecal 100.0% 60.8% 77.1% 75.5% 86.7% R2.64.10 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R2.64.2 Cow Fecal 100.0% 97.9% 100.0% 100.0% 100.0% R2.64.4 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R2.65.4 Cow Fecal 100.0% 100.0% 99.7% 100.0% 100.0% R2.66.10 Cow Fecal 100.0% 60.8% 77.1% 75.5% 86.7% R2.66.4 Cow Fecal 1.9% 0.0% 0.0% 3.7% 5.4% R2.67.3 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R2.67.5 Cow Fecal 100.0% 100.0% 100.0% 99.7% 99.7% R2.68.2 Cow Fecal 100.0% 0.0% 0.0% 0.0% 0.0% R2.68.3 Cow Fecal 89.1% 68.3% 79.4% 84.9% 78.4% R2.68.5 Cow Fecal 100.0% 97.9% 100.0% 100.0% 100.0% R2.68.7 Cow Fecal 81.8% 78.8% 80.6% 81.3% 82.4% R2.69.10 Cow Fecal 100.0% 99.2% 100.0% 99.7% 100.0% R2.69.2 Cow Fecal 100.0% 99.3% 100.0% 99.6% 100.0% R2.70.3 Cow Fecal 3.3% 1.5% 3.7% 4.3% 2.7% R2.70.7 Cow Fecal 73.6% 59.0% 51.3% 62.8% 15.9% R2.70.9 Cow Fecal 100.0% 99.6% 100.0% 100.0% 100.0% R3.1.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R3.11.1 Cow Fecal 100.0% 100.0% 74.2% 100.0% 100.0% R3.11.4 Cow Fecal 91.3% 84.5% 86.3% 86.6% 92.5% R3.11.8 Cow Fecal 84.7% 72.0% 81.1% 82.5% 83.0% R3.11.9 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R3.12.1 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R3.13.2 Cow Fecal 18.4% 18.5% 18.7% 15.5% 17.7% R3.15.2 Cow Fecal 21.2% 17.1% 18.9% 22.8% 21.9% R3.16.4 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R3.19.1 Cow Fecal 30.1% 27.6% 28.2% 34.6% 28.5% R3.19.5 Cow Fecal 51.7% 49.4% 46.5% 52.4% 47.6% R3.2.2 Cow Fecal 56.3% 48.5% 51.3% 52.8% 52.5% R3.21.5 Cow Fecal 62.4% 33.4% 52.1% 24.6% 53.8% R3.22.1 Cow Fecal 99.8% 100.0% 100.0% 100.0% 100.0% R3.23.1 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R3.23.5 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R3.23.8 Cow Fecal 100.0% 95.1% 98.8% 96.9% 98.8% R3.23.9 Cow Fecal 32.8% 24.8% 100.0% 24.8% 22.1% R3.25.7 Cow Fecal 100.0% 84.6% 100.0% 89.6% 100.0% R3.28.6 Cow Fecal 23.1% 19.2% 21.2% 24.6% 23.6% R3.28.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R3.29.1 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R3.30.2 Cow Fecal 100.0% 95.1% 99.0% 96.8% 99.7% R3.32.2 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R3.32.5 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R3.32.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R3.41.2 Cow Fecal 69.5% 41.0% 41.6% 93.7% 61.8% R3.41.4 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R3.42.2 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R3.42.3 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R3.43.2 Cow Fecal 100.0% 43.9% 71.3% 67.8% 70.7% R3.44.1 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R3.44.5 Cow Fecal 76.9% 58.7% 62.7% 60.8% 72.4% R3.45.5 Cow Fecal 92.4% 87.1% 91.0% 91.4% 95.8% R3.47.1 Cow Fecal 0.0% 0.0% 0.0% 0.0% 0.0% R3.48.2 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R3.48.9 Cow Fecal 69.3% 61.9% 67.3% 70.8% 68.1% R3.49.4 Cow Fecal 38.8% 7.6% 26.5% 0.0% 26.7% R3.51.1 Cow Fecal 99.6% 4.1% 99.9% 0.0% 99.3% R3.51.4 Cow Fecal 98.8% 98.8% 98.8% 99.5% 99.0% R3.52.5 Cow Fecal 100.0% 100.0% 100.0% 100.0% 98.8% R3.52.7 Cow Fecal 97.5% 94.0% 95.1% 95.2% 96.8% R3.54.4 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R3.57.5 Cow Fecal 100.0% 98.4% 100.0% 99.5% 100.0% R3.58.3 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R3.58.4 Cow Fecal 68.4% 59.6% 61.1% 61.3% 64.9% R3.6.5 Cow Fecal 47.2% 25.9% 54.1% 19.4% 44.4% R3.60.3 Cow Fecal 99.9% 100.0% 100.0% 100.0% 99.9% R3.7.1 Cow Fecal 100.0% 100.0% 100.0% 99.7% 100.0% R3.7.10 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R3.7.4 Cow Fecal 100.0% 100.0% 100.0% 99.5% 100.0% R3.7.9 Cow Fecal 100.0% 96.7% 98.4% 97.6% 98.6% R3.8.1 Cow Fecal 100.0% 35.5% 40.3% 11.5% 17.8% R3.8.3 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R5.12.10 Cow Fecal 95.8% 7.3% 100.0% 2.0% 94.4% R5.12.3 Cow Fecal 100.0% 100.0% 100.0% 98.4% 100.0% R5.12.6 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R5.13.4 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R5.13.5 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R5.13.9 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R5.14.2 Cow Fecal 84.4% 44.0% 91.8% 10.1% 100.0% R5.18.4 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R5.21.5 Cow Fecal 18.2% 20.5% 23.8% 20.7% 17.7% R5.22.9 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R5.24.2 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R5.3.3 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R5.3.5 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R5.3.6 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R5.3.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R5.35.3 Cow Fecal 4.7% 17.6% 4.6% 24.5% 18.0% R5.35.4 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R5.36.7 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R5.4.1 Cow Fecal 100.0% 100.0% 100.0% 97.9% 100.0% R5.4.5 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R5.40.5 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R5.49.2 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R5.49.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R5.5.2 Cow Fecal 100.0% 96.6% 100.0% 100.0% 100.0% R5.55.1 Cow Fecal 100.0% 100.0% 100.0% 97.9% 100.0% R5.55.2 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R5.58.10 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R5.58.5 Cow Fecal 100.0% 100.0% 100.0% 92.5% 100.0% R5.58.7 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R5.58.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R5.6.10 Cow Fecal 100.0% 100.0% 100.0% 100.0% 98.6% R5.6.5 Cow Fecal 99.7% 99.3% 100.0% 100.0% 100.0% R5.60.4 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R5.7.3 Cow Fecal 100.0% 68.5% 100.0% 90.4% 99.6% R5.8.10 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R5.8.4 Cow Fecal 100.0% 100.0% 100.0% 85.8% 100.0% R5.8.6 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R7.1.10 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R7.1.2 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R7.10.7 Calf Fecal 99.3% 92.7% 91.6% 100.0% 96.0% R7.11.8 Calf Fecal 100.0% 51.7% 88.4% 56.8% 98.4% R7.12.7 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R7.12.8 Calf Fecal 98.3% 99.3% 100.0% 99.4% 99.9% R7.13.3 Calf Fecal 100.0% 100.0% 96.9% 100.0% 100.0% R7.13.9 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R7.14.5 Calf Fecal 100.0% 15.9% 99.8% 7.6% 100.0% R7.14.6 Calf Fecal 62.7% 16.7% 9.0% 33.6% 29.4% R7.15.1 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R7.15.2 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R7.15.5 Calf Fecal 100.0% 99.7% 100.0% 99.3% 100.0% R7.17.9 Cow Fecal 100.0% 100.0% 100.0% 69.8% 99.6% R7.19.2 Cow Fecal 98.1% 98.5% 97.2% 99.3% 78.4% R7.2.1 Calf Fecal 58.2% 36.2% 9.8% 70.5% 0.0% R7.2.5 Calf Fecal 79.8% 55.5% 19.1% 93.3% 0.0% R7.2.6 Calf Fecal 100.0% 91.9% 90.6% 84.2% 100.0% R7.2.7 Calf Fecal 100.0% 78.5% 89.6% 74.7% 100.0% R7.21.5 Cow Fecal 100.0% 100.0% 100.0% 100.0% 87.7% R7.23.4 Cow Fecal 98.8% 100.0% 70.9% 100.0% 77.6% R7.26.10 Cow Fecal 100.0% 100.0% 83.2% 100.0% 98.9% R7.27.10 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R7.3.2 Calf Fecal 100.0% 100.0% 100.0% 70.5% 100.0% R7.30.1 Cow Fecal 98.4% 97.8% 93.2% 98.5% 79.0% R7.34.9 Cow Fecal 98.5% 98.6% 96.9% 98.4% 72.8% R7.39.3 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R7.4.2 Calf Fecal 100.0% 54.0% 97.9% 51.8% 100.0% R7.47.2 Cow Fecal 100.0% 100.0% 100.0% 100.0% 66.2% R7.48.1 Cow Fecal 100.0% 0.0% 0.0% 0.0% 100.0% R7.49.2 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R7.5.6 Calf Fecal 28.9% 18.4% 19.5% 7.4% 21.6% R7.50.4 Cow Fecal 99.9% 78.2% 100.0% 99.5% 99.3% R7.50.5 Cow Fecal 9.4% 10.0% 13.7% 16.1% 9.8% R7.51.1 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R7.51.5 Cow Fecal 100.0% 100.0% 100.0% 100.9% 100.0% R7.54.4 Cow Fecal 99.7% 100.0% 100.0% 100.0% 74.7% R7.55.2 Cow Fecal 84.9% 78.6% 31.0% 100.0% 0.0% R7.55.3 Cow Fecal 100.0% 100.0% 99.9% 99.7% 99.3% R7.55.9 Cow Fecal 100.0% 99.5% 99.5% 99.8% 100.0% R7.59.9 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R7.64.2 Cow Fecal 100.0% 100.0% 99.9% 100.0% 100.0% R7.65.1 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R7.67.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R7.68.1 Cow Fecal 53.6% 83.3% 2.1% 100.0% 0.0% R7.68.3 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R7.69.1 Cow Fecal 100.0% 99.7% 99.3% 100.0% 38.9% R7.72.3 Cow Fecal 100.0% 100.0% 100.0% 99.9% 81.5% R7.73.2 Cow Fecal 88.0% 100.0% 48.4% 100.0% 81.6% R7.83.1 Cow Fecal 100.0% 100.0% 93.9% 100.0% 31.6% R17.1.2 Cow Fecal 97.8% 99.6% 90.2% 100.0% 100.0% R17.10.3 Cow Fecal 40.5% 37.3% 52.7% 53.0% 48.0% R17.10.4 Cow Fecal 100.0% 100.0% 24.1% 100.0% 98.9% R17.10.5 Cow Fecal 43.5% 57.9% 21.0% 20.5% 21.2% R17.11.10 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R17.11.2 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R17.13.8 Cow Fecal 67.9% 73.2% 72.2% 100.0% 31.9% R17.14.10 Cow Fecal 99.2% 100.0% 82.1% 78.6% 94.9% R17.14.2 Cow Fecal 34.9% 96.2% 37.9% 34.1% 57.8% R17.15.1 Cow Fecal 99.8% 99.3% 100.0% 100.0% 100.0% R17.17.1 Cow Fecal 100.0% 100.0% 100.0% 99.9% 100.0% R17.2.2 Cow Fecal 46.8% 74.0% 77.8% 85.7% 58.2% R17.2.4 Cow Fecal 99.9% 100.0% 78.1% 65.3% 77.3% R17.2.7 Cow Fecal 100.0% 100.0% 36.6% 100.0% 100.0% R17.2.9 Cow Fecal 59.9% 76.3% 2.6% 24.7% 35.9% R17.20.2 Cow Fecal 100.0% 100.0% 98.8% 96.0% 100.0% R17.21.1 Cow Fecal 100.0% 100.0% 99.9% 99.7% 100.0% R17.22.4 Cow Fecal 100.0% 100.0% 100.0% 100.0% 57.0% R17.22.5 Cow Fecal 100.0% 67.7% 88.9% 69.1% 59.2% R17.23.1 Cow Fecal 97.6% 100.0% 37.1% 81.3% 94.7% R17.23.3 Cow Fecal 68.8% 65.1% 71.6% 60.1% 60.4% R17.23.5 Cow Fecal 100.0% 96.2% 90.1% 92.6% 100.0% R17.23.6 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R17.25.6 Cow Fecal 99.3% 100.0% 79.9% 95.5% 98.3% R17.25.9 Cow Fecal 71.6% 79.7% 89.3% 78.6% 85.9% R17.26.5 Cow Fecal 100.0% 87.3% 100.0% 0.0% 0.0% R17.26.6 Cow Fecal 35.2% 100.0% 100.0% 99.4% 99.7% R17.26.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R17.28.2 Cow Fecal 57.5% 48.4% 41.7% 58.8% 59.2% R17.28.3 Cow Fecal 91.9% 78.8% 91.6% 77.5% 83.0% R17.28.6 Cow Fecal 95.2% 79.6% 96.0% 64.1% 95.9% R17.29.1 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R17.29.2 Cow Fecal 24.0% 24.5% 28.7% 17.7% 26.2% R17.29.4 Cow Fecal 95.2% 99.0% 83.8% 43.9% 91.7% R17.29.5 Cow Fecal 96.9% 100.0% 44.2% 52.3% 42.2% R17.29.6 Cow Fecal 96.3% 99.8% 98.3% 100.0% 100.0% R17.29.8 Cow Fecal 77.9% 82.8% 75.2% 77.0% 78.5% R17.29.9 Cow Fecal 0.0% 0.0% 0.0% 0.0% 0.0% R17.30.1 Cow Fecal 93.0% 92.9% 23.1% 40.2% 88.7% R17.30.7 Cow Fecal 77.4% 85.6% 78.3% 51.8% 78.3% R17.31.4 Cow Fecal 19.4% 13.9% 20.7% 11.9% 15.0% R17.31.5 Cow Fecal 100.0% 99.9% 100.0% 99.4% 100.0% R17.31.7 Cow Fecal 80.8% 97.9% 66.9% 84.0% 87.6% R17.32.3 Cow Fecal 41.0% 68.8% 77.9% 81.1% 75.3% R17.32.5 Cow Fecal 100.0% 100.0% 73.4% 93.4% 100.0% R17.32.6 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R17.33.2 Cow Fecal 100.0% 77.8% 19.2% 100.0% 100.0% R17.33.6 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R17.34.3 Cow Fecal 42.4% 99.5% 99.6% 98.8% 99.8% R17.35.10 Cow Fecal 39.0% 37.0% 38.6% 39.1% 39.6% R17.35.2 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R17.35.4 Cow Fecal 47.9% 48.4% 50.7% 47.7% 49.5% R17.35.9 Cow Fecal 100.0% 100.0% 72.6% 95.1% 100.0% R17.36.10 Cow Fecal 59.2% 87.7% 31.7% 79.2% 62.7% R17.36.3 Cow Fecal 49.3% 38.3% 37.5% 34.1% 35.4% R17.36.7 Cow Fecal 89.8% 98.7% 69.0% 78.8% 86.7% R17.38.5 Cow Fecal 74.2% 66.8% 84.6% 58.6% 88.5% R17.38.6 Cow Fecal 100.0% 100.0% 100.0% 29.2% 100.0% R17.38.9 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R17.39.2 Cow Fecal 41.6% 39.9% 42.7% 40.7% 44.1% R17.39.8 Cow Fecal 57.1% 100.0% 0.0% 14.4% 88.6% R17.40.9 Cow Fecal 0.0% 0.0% 0.0% 0.0% 1.7% R17.41.2 Cow Fecal 0.0% 0.0% 0.0% 0.0% 0.0% R17.43.2 Cow Fecal 50.1% 86.1% 15.3% 24.7% 15.2% R17.43.3 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R17.44.8 Cow Fecal 50.7% 22.0% 100.0% 99.7% 21.7% R17.47.1 Cow Fecal 98.2% 91.0% 99.4% 93.4% 100.0% R17.48.2 Cow Fecal 99.5% 99.8% 99.6% 99.9% 100.0% R17.49.8 Cow Fecal 86.5% 99.8% 99.8% 100.0% 99.9% R17.51.6 Cow Fecal 99.7% 81.7% 100.0% 100.0% 100.0% R17.54.6 Cow Fecal 0.0% 0.0% 0.0% 0.0% 0.0% R17.58.4 Cow Fecal 100.0% 100.0% 97.1% 99.5% 100.0% R17.58.7 Cow Fecal 61.6% 98.9% 54.7% 54.8% 85.9% R17.60.2 Cow Fecal 93.4% 100.0% 100.0% 81.9% 78.5% R17.60.3 Cow Fecal 53.0% 37.8% 23.7% 44.4% 45.6% R17.60.5 Cow Fecal 53.6% 74.6% 73.4% 61.2% 56.9% R17.64.10 Cow Fecal 90.3% 88.3% 0.0% 46.9% 54.7% R17.64.9 Cow Fecal 81.0% 85.7% 57.3% 62.0% 51.4% R17.67.9 Cow Fecal 13.0% 51.7% 0.0% 11.8% 10.9% R17.68.2 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R17.68.9 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R17.7.2 Cow Fecal 90.0% 94.7% 93.9% 85.2% 89.8% R17.7.7 Cow Fecal 100.0% 100.0% 80.8% 66.2% 82.9% R17.7.9 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R17.8.2 Cow Fecal 66.3% 74.3% 71.3% 71.3% 71.3% R17.8.9 Cow Fecal 0.0% 84.6% 72.2% 0.0% 0.0% R18.1.2 Cow Fecal 38.5% 90.6% 33.8% 38.3% 39.3% R18.1.3 Cow Fecal 0.0% 57.0% 0.0% 0.0% 0.0% R18.1.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.1.10 Cow Fecal 97.6% 87.3% 52.1% 82.7% 84.2% R18.2.4 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.2.6 Cow Fecal 88.1% 69.2% 55.1% 84.4% 74.8% R18.2.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.3.3 Cow Fecal 99.8% 100.0% 100.0% 100.0% 100.0% R18.5.2 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.7.7 Cow Fecal 99.0% 100.0% 96.6% 100.0% 100.0% R18.8.3 Cow Fecal 81.1% 56.9% 35.6% 60.8% 70.8% R18.8.4 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.8.5 Cow Fecal 24.0% 49.3% 74.4% 43.0% 73.1% R18.8.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.10.3 Cow Fecal 83.2% 60.8% 100.0% 100.0% 63.3% R18.10.10 Cow Fecal 99.8% 100.0% 100.0% 61.2% 100.0% R18.11.4 Cow Fecal 99.7% 99.8% 100.0% 100.0% 100.0% R18.13.6 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.13.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.16.4 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.16.5 Cow Fecal 4.4% 11.7% 9.1% 10.8% 8.4% R18.17.3 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.18.5 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.18.7 Cow Fecal 70.6% 79.4% 6.4% 8.8% 26.1% R18.18.9 Cow Fecal 100.0% 99.7% 100.0% 100.0% 100.0% R18.18.10 Cow Fecal 100.0% 56.9% 100.0% 91.1% 74.0% R18.20.3 Cow Fecal 96.9% 100.0% 44.2% 52.3% 42.2% R18.20.4 Cow Fecal 100.0% 100.0% 100.0% 82.5% 80.8% R18.21.3 Cow Fecal 69.6% 65.4% 32.1% 34.4% 3.8% R18.21.6 Cow Fecal 31.9% 17.1% 12.5% 0.0% 3.6% R18.21.9 Cow Fecal 57.1% 100.0% 0.0% 14.4% 88.7% R18.21.10 Cow Fecal 48.1% 51.0% 34.1% 0.0% 44.6% R18.22.8 Cow Fecal 36.5% 64.4% 41.5% 59.0% 96.4% R18.23.1 Cow Fecal 99.7% 97.7% 80.1% 58.3% 57.3% R18.23.2 Cow Fecal 19.4% 66.5% 0.0% 0.4% 6.7% R18.25.1 Cow Fecal 70.8% 67.8% 70.7% 49.8% 51.6% R18.25.2 Cow Fecal 93.2% 99.7% 100.0% 85.7% 79.2% R18.25.3 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.25.5 Cow Fecal 46.5% 48.1% 100.0% 45.4% 35.2% R18.25.6 Cow Fecal 96.0% 83.1% 65.6% 88.6% 77.2% R18.26.2 Cow Fecal 100.0% 81.7% 75.4% 32.0% 79.6% R18.26.4 Cow Fecal 96.3% 72.6% 58.9% 31.0% 76.0% R18.26.7 Cow Fecal 99.7% 100.0% 100.0% 100.0% 100.0% R18.26.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.27.2 Cow Fecal 63.3% 84.6% 40.8% 60.2% 48.3% R18.27.5 Cow Fecal 100.0% 30.1% 42.1% 14.8% 16.1% R18.27.7 Cow Fecal 100.0% 100.0% 100.0% 61.5% 100.0% R18.28.1 Cow Fecal 42.3% 47.9% 34.3% 13.2% 11.9% R18.28.8 Cow Fecal 87.3% 100.0% 100.0% 52.4% 76.0% R18.28.9 Cow Fecal 100.0% 99.9% 100.0% 100.0% 100.0% R18.29.1 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.29.2 Cow Fecal 93.8% 76.0% 100.0% 98.8% 96.5% R18.29.3 Cow Fecal 30.3% 21.9% 23.7% 9.3% 22.9% R18.30.2 Cow Fecal 100.0% 98.0% 100.0% 88.7% 100.0% R18.30.8 Cow Fecal 0.0% 100.0% 100.0% 79.1% 93.9% R18.31.2 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.31.3 Cow Fecal 100.0% 100.0% 100.0% 60.5% 100.0% R18.31.6 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.31.7 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.31.9 Cow Fecal 82.8% 75.2% 48.5% 59.7% 49.8% R18.31.10 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.32.2 Cow Fecal 16.4% 11.2% 9.8% 2.4% 15.3% R18.32.3 Cow Fecal 100.0% 100.0% 96.9% 100.0% 96.7% R18.34.3 Cow Fecal 19.3% 18.8% 23.8% 27.6% 51.8% R18.36.3 Cow Fecal 56.5% 20.3% 41.5% 42.8% 49.0% R18.36.5 Cow Fecal 100.0% 100.0% 98.2% 52.6% 98.7% R18.36.8 Cow Fecal 99.5% 100.0% 93.3% 98.9% 100.0% R18.37.2 Cow Fecal 100.0% 99.8% 100.0% 100.0% 99.5% R18.37.8 Cow Fecal 69.6% 47.7% 90.4% 63.2% 74.8% R18.37.10 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.39.1 Cow Fecal 28.4% 29.1% 25.1% 27.1% 26.8% R18.39.8 Cow Fecal 100.0% 57.7% 75.5% 45.7% 67.6% R18.39.10 Cow Fecal 16.2% 19.2% 17.5% 17.3% 16.7% R18.41.5 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.41.8 Cow Fecal 23.7% 57.8% 64.7% 31.8% 39.2% R18.42.6 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.42.7 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.43.3 Cow Fecal 100.0% 100.0% 100.0% 74.3% 88.7% R18.44.6 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.45.6 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.46.8 Cow Fecal 73.3% 99.1% 37.3% 42.4% 21.1% R18.46.10 Cow Fecal 70.5% 100.0% 45.5% 46.2% 60.3% R18.48.1 Cow Fecal 68.0% 100.0% 38.6% 35.8% 31.2% R18.48.2 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.48.7 Cow Fecal 100.0% 99.9% 99.9% 100.0% 100.0% R18.48.9 Cow Fecal 74.4% 79.0% 62.4% 41.8% 42.2% R18.49.7 Cow Fecal 100.0% 96.1% 100.0% 0.0% 0.0% R18.50.3 Cow Fecal 79.4% 100.0% 100.0% 100.0% 100.0% R18.50.5 Cow Fecal 31.1% 30.8% 27.1% 28.0% 28.0% R18.51.6 Cow Fecal 45.1% 51.6% 45.1% 40.5% 44.6% R18.52.1 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.53.3 Cow Fecal 75.9% 51.5% 100.0% 50.6% 41.9% R18.53.5 Cow Fecal 51.4% 41.4% 20.1% 26.0% 3.6% R18.55.6 Cow Fecal 73.1% 46.0% 28.9% 28.1% 29.7% R18.55.8 Cow Fecal 63.8% 54.2% 1.4% 0.0% 0.0% R18.56.1 Cow Fecal 96.6% 92.6% 61.8% 61.0% 85.1% R18.56.4 Cow Fecal 98.2% 91.0% 99.4% 93.4% 100.0% R18.56.9 Cow Fecal 64.4% 96.7% 48.5% 43.1% 60.9% R18.58.1 Cow Fecal 98.8% 99.8% 99.2% 99.7% 100.0% R18.58.2 Cow Fecal 100.0% 100.0% 36.6% 100.0% 100.0% R18.58.9 Cow Fecal 80.1% 64.2% 36.0% 65.6% 70.0% R18.58.10 Cow Fecal 100.0% 95.4% 94.5% 0.0% 0.0% R18.60.1 Cow Fecal 99.1% 99.8% 99.0% 99.6% 98.8% R18.60.3 Cow Fecal 80.7% 100.0% 100.0% 66.3% 53.8% R18.60.4 Cow Fecal 19.0% 15.1% 20.8% 14.4% 19.6% R18.60.5 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.60.10 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R18.61.7 Cow Fecal 3.8% 1.3% 10.1% 0.8% 0.7% R19.1.5 Cow Fecal 86.5% 100.0% 54.8% 23.6% 69.1% R19.10.7 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.12.5 Cow Fecal 15.0% 14.8% 32.5% 15.0% 5.1% R19.13.1 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.13.10 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.15.3 Cow Fecal 100.0% 100.0% 40.8% 68.0% 98.5% R19.15.4 Cow Fecal 43.4% 66.4% 17.7% 71.2% 5.1% R19.15.6 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.15.7 Cow Fecal 30.4% 11.6% 19.8% 24.7% 28.2% R19.15.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.15.9 Cow Fecal 100.0% 100.0% 64.9% 37.6% 94.2% R19.16.9 Cow Fecal 100.0% 100.0% 66.8% 35.9% 84.6% R19.17.2 Cow Fecal 15.4% 16.9% 28.9% 17.9% 20.4% R19.17.8 Cow Fecal 56.5% 22.4% 48.5% 23.7% 29.3% R19.18.1 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.23.3 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.23.4 Cow Fecal 100.0% 100.0% 41.8% 100.0% 100.0% R19.23.8 Cow Fecal 62.1% 87.2% 68.5% 38.9% 52.1% R19.24.1 Cow Fecal 97.5% 100.0% 83.0% 100.0% 100.0% R19.24.2 Cow Fecal 84.7% 15.9% 64.0% 83.2% 74.1% R19.26.2 Cow Fecal 40.1% 85.7% 39.6% 102.5% 38.8% R19.27.10 Cow Fecal 36.7% 100.0% 39.8% 41.8% 41.6% R19.27.5 Cow Fecal 68.6% 100.0% 63.2% 45.0% 56.8% R19.27.7 Cow Fecal 100.0% 70.6% 94.0% 100.0% 34.7% R19.28.10 Cow Fecal 100.0% 100.0% 100.0% 100.0% 82.1% R19.28.5 Cow Fecal 95.7% 47.1% 100.0% 80.5% 45.7% R19.28.6 Cow Fecal 100.0% 100.0% 83.8% 85.9% 88.5% R19.29.3 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.29.4 Cow Fecal 100.0% 100.0% 75.2% 81.4% 20.3% R19.29.5 Cow Fecal 100.0% 100.0% 67.5% 51.3% 82.7% R19.29.7 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.29.9 Cow Fecal 97.9% 54.5% 66.5% 36.6% 87.9% R19.3.3 Cow Fecal 100.0% 100.0% 74.0% 99.2% 88.6% R19.30.1 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.30.11 Cow Fecal 100.0% 100.0% 95.0% 89.2% 99.0% R19.30.2 Cow Fecal 92.5% 94.5% 72.2% 84.6% 57.6% R19.30.5 Cow Fecal 58.7% 16.8% 73.6% 64.9% 37.5% R19.30.6 Cow Fecal 100.0% 100.0% 33.7% 83.0% 40.0% R19.31.10 Cow Fecal 99.9% 72.2% 97.9% 61.8% 94.1% R19.31.8 Cow Fecal 17.4% 49.0% 66.3% 50.2% 67.6% R19.34.1 Cow Fecal 85.0% 100.0% 75.4% 93.3% 99.1% R19.35.10 Cow Fecal 83.9% 76.8% 81.1% 52.4% 83.3% R19.35.5 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.35.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.35.9 Cow Fecal 100.0% 55.1% 100.0% 91.5% 96.5% R19.36.4 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.36.5 Cow Fecal 99.4% 90.1% 100.0% 100.0% 100.0% R19.37.1 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.37.10 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.37.2 Cow Fecal 98.0% 100.0% 100.0% 23.6% 97.9% R19.37.6 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.38.10 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.38.2 Cow Fecal 100.0% 100.0% 95.2% 16.6% 83.6% R19.38.3 Cow Fecal 13.4% 0.0% 0.0% 0.0% 0.0% R19.38.6 Cow Fecal 74.8% 65.6% 0.0% 97.9% 98.5% R19.38.7 Cow Fecal 25.2% 1.7% 0.0% 0.0% 0.0% R19.39.1 Cow Fecal 98.9% 100.0% 100.0% 100.0% 101.3% R19.39.10 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.39.7 Cow Fecal 99.1% 24.3% 87.5% 76.3% 26.4% R19.39.9 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.40. Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.40.1 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.40.10 Cow Fecal 27.8% 49.9% 33.9% 29.1% 8.6% R19.40.4 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.40.7 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.41.10 Cow Fecal 74.9% 47.9% 51.2% 45.9% 51.3% R19.41.7 Cow Fecal 96.7% 97.0% 86.6% 88.1% 100.0% R19.41.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.42.3 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.42.6 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.43.2 Cow Fecal 62.2% 58.2% 58.8% 51.3% 58.7% R19.44.6 Cow Fecal 0.0% 0.0% 0.0% 0.0% 0.0% R19.46.10 Cow Fecal 72.6% 81.3% 26.0% 43.6% 54.8% R19.46.2 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.46.4 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.47.10 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.47.2 Cow Fecal 100.0% 100.0% 85.5% 100.0% 100.0% R19.47.3 Cow Fecal 34.3% 25.2% 15.0% 16.1% 17.5% R19.47.5 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.48.3 Cow Fecal 0.0% 100.0% 100.0% 100.0% 97.4% R19.48.5 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.48.8 Cow Fecal 100.0% 73.9% 55.9% 31.1% 57.3% R19.49.10 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.49.3 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.49.4 Cow Fecal 48.6% 71.9% 3.8% 18.3% 81.0% R19.49.5 Cow Fecal 0.0% 0.0% 0.0% 0.0% 0.0% R19.49.6 Cow Fecal 100.0% 19.4% 47.3% 43.3% 45.4% R19.49.9 Cow Fecal 36.3% 26.8% 26.9% 20.4% 22.5% R19.5.6 Cow Fecal 99.5% 46.7% 0.0% 19.5% 41.9% R19.50.2 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.50.4 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.51.1 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.51.3 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.52.2 Cow Fecal 85.1% 99.2% 48.4% 22.7% 76.8% R19.52.3 Cow Fecal 100.0% 100.0% 100.0% 97.7% 100.0% R19.52.5 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.52.9 Cow Fecal 76.3% 48.6% 49.3% 40.5% 26.7% R19.53.1 Cow Fecal 100.0% 100.0% 100.0% 100.0% 97.4% R19.53.10 Cow Fecal 99.9% 99.1% 70.9% 41.9% 93.8% R19.53.8 Cow Fecal 65.0% 80.7% 58.9% 32.0% 43.2% R19.54.1 Cow Fecal 46.6% 27.2% 20.8% 33.4% 3.5% R19.54.7 Cow Fecal 55.0% 0.0% 0.0% 10.8% 0.0% R19.55.2 Cow Fecal 79.5% 36.8% 38.1% 39.4% 34.8% R19.55.8 Cow Fecal 47.7% 51.4% 51.1% 46.5% 27.7% R19.56.10 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.56.4 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.56.7 Cow Fecal 70.7% 92.4% 61.7% 70.6% 60.3% R19.57.10 Cow Fecal 27.0% 29.6% 24.2% 24.2% 22.7% R19.57.2 Cow Fecal 62.9% 89.2% 79.5% 76.9% 77.6% R19.57.5 Cow Fecal 76.2% 75.0% 0.0% 64.8% 29.8% R19.58.10 Cow Fecal 53.7% 49.4% 35.5% 26.8% 46.1% R19.58.7 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.58.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.59.7 Cow Fecal 76.5% 50.1% 45.9% 28.1% 22.8% R19.59.9 Cow Fecal 73.2% 58.9% 50.9% 29.4% 55.6% R19.60.10 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.60.12 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.60.14 Cow Fecal 100.0% 100.0% 78.8% 56.2% 67.2% R19.62.4 Cow Fecal 31.9% 27.3% 30.6% 32.3% 27.2% R19.62.7 Cow Fecal 37.0% 37.2% 25.8% 50.1% 10.8% R19.62.9 Cow Fecal 100.0% 83.1% 38.2% 27.1% 58.4% R19.63.3 Cow Fecal 67.2% 75.3% 54.6% 39.5% 45.6% R19.63.4 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.63.7 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.64.3 Cow Fecal 98.0% 94.0% 10.5% 10.2% 31.8% R19.65.1 Cow Fecal 14.0% 2.2% 20.6% 0.0% 30.3% R19.65.10 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.65.2 Cow Fecal 47.9% 45.7% 48.6% 45.0% 46.0% R19.65.5 Cow Fecal 31.4% 31.0% 32.9% 32.0% 31.7% R19.65.6 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.66.10 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.66.8 Cow Fecal 88.9% 71.0% 69.4% 92.5% 85.4% R19.7.9 Cow Fecal 100.0% 76.1% 93.9% 100.0% 59.8% R19.70.6 Cow Fecal 54.5% 48.2% 58.9% 53.6% 53.6% R19.70.8 Cow Fecal 91.2% 93.6% 55.3% 2.8% 33.3% R19.9.3 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R19.9.9 Cow Fecal 100.0% 67.2% 49.8% 24.2% 75.9% R23.10.4 Cow Fecal 100.0% 3.1% 99.8% 97.2% 99.6% R23.10.8 Cow Fecal 99.7% 99.6% 99.5% 99.7% 99.6% R23.11.2 Cow Fecal 59.6% 0.0% 1.3% 17.6% 40.7% R23.11.9 Cow Fecal 77.5% 10.2% 2.3% 61.1% 65.0% R23.12.2 Cow Fecal 0.8% 0.0% 0.0% 14.1% 10.5% R23.13.1 Cow Fecal 99.9% 99.7% 99.4% 99.7% 99.7% R23.14.5 Cow Fecal 74.5% 75.2% 71.4% 75.2% 75.2% R23.15.1 Cow Fecal 16.8% 79.5% 94.5% 94.9% 57.3% R23.15.10 Cow Fecal 99.8% 100.0% 100.0% 99.5% 99.6% R23.17.5 Cow Fecal 96.7% 97.8% 99.2% 97.3% 98.0% R23.18.2 Cow Fecal 88.9% 100.5% 100.5% 99.8% 100.5% R23.18.5 Cow Fecal 99.8% 99.8% 99.7% 99.7% 99.7% R23.18.9 Cow Fecal 99.6% 51.7% 99.2% 82.1% 99.4% R23.22.9 Cow Fecal 99.1% 99.4% 99.1% 98.9% 99.1% R23.23.8 Cow Fecal 99.3% 99.1% 98.9% 99.1% 99.1% R23.24.1 Cow Fecal 99.1% 99.1% 98.9% 99.3% 99.3% R23.24.3 Cow Fecal 99.0% 99.3% 98.8% 99.3% 99.3% R23.25.3 Cow Fecal 95.0% 94.7% 81.1% 95.7% 94.4% R23.28.6 Cow Fecal 99.7% 100.0% 51.8% 97.4% 99.8% R23.28.8 Cow Fecal 98.0% 97.9% 94.6% 97.7% 97.6% R23.3.5 Cow Fecal 89.1% 0.0% 0.0% 80.1% 11.9% R23.31.6 Cow Fecal 99.4% 99.6% 99.6% 99.0% 99.6% R23.32.1 Cow Fecal 99.6% 73.2% 98.4% 99.3% 99.3% R23.32.5 Cow Fecal 24.3% 5.7% 47.1% 11.7% 2.7% R23.32.6 Cow Fecal 95.8% 92.1% 92.7% 92.9% 97.3% R23.33.5 Cow Fecal 99.7% 100.0% 99.3% 99.9% 99.1% R23.34.4 Cow Fecal 99.6% 90.2% 99.6% 22.8% 99.7% R23.35.1 Cow Fecal 99.0% 99.4% 99.1% 99.4% 99.4% R23.35.10 Cow Fecal 99.4% 99.2% 82.9% 99.7% 99.1% R23.35.4 Cow Fecal 99.4% 99.7% 99.1% 99.7% 99.3% R23.35.8 Cow Fecal 99.5% 99.9% 98.5% 98.6% 99.2% R23.36.1 Cow Fecal 22.1% 25.3% 63.2% 0.0% 69.5% R23.36.3 Cow Fecal 99.2% 100.1% 97.5% 99.7% 99.4% R23.36.4 Cow Fecal 99.5% 100.0% 98.1% 0.0% 99.3% R23.36.6 Cow Fecal 97.7% 99.6% 98.3% 97.5% 99.0% R23.39.7 Cow Fecal 99.5% 99.6% 99.5% 99.8% 99.8% R23.4.7 Cow Fecal 99.7% 99.8% 99.6% 99.5% 99.6% R23.40.6 Cow Fecal 99.6% 99.9% 12.5% 99.6% 99.4% R23.41.10 Cow Fecal 91.8% 30.9% 14.5% 92.9% 86.6% R23.42.8 Cow Fecal 68.1% 0.0% 0.0% 44.1% 13.6% R23.44.10 Cow Fecal 99.5% 100.0% 99.7% 98.1% 99.7% R23.44.4 Cow Fecal 94.8% 19.5% 95.1% 91.2% 0.0% R23.44.6 Cow Fecal 99.9% 97.2% 98.2% 96.9% 98.1% R23.44.8 Cow Fecal 95.0% 84.5% 84.5% 95.9% 96.3% R23.45.2 Cow Fecal 76.8% 76.8% 87.1% 76.8% 84.9% R23.45.9 Cow Fecal 99.3% 100.0% 99.6% 98.1% 98.8% R23.48.3 Cow Fecal 97.7% 91.6% 86.4% 94.4% 92.4% R23.48.6 Cow Fecal 7.6% 10.9% 0.0% 0.0% 20.6% R23.49.1 Cow Fecal 95.1% 97.9% 95.3% 96.9% 98.3% R23.49.4 Cow Fecal 98.5% 94.7% 93.0% 96.0% 97.0% R23.49.5 Cow Fecal 82.0% 99.8% 84.4% 99.9% 51.2% R23.5.5 Cow Fecal 86.0% 76.4% 95.1% 85.5% 93.1% R23.50.3 Cow Fecal 96.5% 97.6% 98.5% 97.2% 97.8% R23.51.1 Cow Fecal 99.5% 99.1% 95.1% 97.9% 99.0% R23.51.10 Cow Fecal 27.5% 0.0% 100.0% 14.7% 24.6% R23.51.8 Cow Fecal 99.8% 95.4% 87.4% 96.6% 98.9% R23.52.3 Cow Fecal 87.7% 74.5% 77.2% 80.0% 87.1% R23.53.8 Cow Fecal 6.3% 0.0% 0.0% 0.0% 23.8% R23.56.5 Cow Fecal 64.8% 99.8% 81.8% 0.0% 10.9% R23.56.9 Cow Fecal 99.5% 76.2% 45.0% 21.2% 93.3% R23.57.1 Cow Fecal 0.0% 0.0% 0.0% 0.0% 12.9% R23.58.2 Cow Fecal 100.0% 0.0% 99.9% 99.8% 99.8% R23.60.3 Cow Fecal 55.3% 86.6% 99.2% 81.0% 90.5% R23.61.1 Cow Fecal 1.9% 0.0% 99.6% 99.6% 0.0% R23.61.3 Cow Fecal 99.8% 99.8% 100.0% 99.7% 99.3% R23.64.7 Cow Fecal 0.0% 0.0% 4.1% 0.0% 23.7% R23.66.2 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R23.66.7 Cow Fecal 15.6% 0.0% 69.1% 21.0% 36.3% R23.66.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R23.67.9 Cow Fecal 28.1% 0.0% 38.9% 24.1% 33.8% R23.68.10 Cow Fecal 96.8% 96.8% 96.8% 96.8% 96.8% R23.8.8 Cow Fecal 99.4% 99.4% 99.1% 99.3% 99.5% R23.9.7 Cow Fecal 99.3% 99.5% 99.3% 99.4% 99.1% R24.1.1 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R24.1.6 Cow Fecal 99.8% 100.0% 100.0% 100.0% 99.9% R24.10.1 Cow Fecal 69.8% 71.1% 64.4% 62.2% 69.6% R24.10.10 Cow Fecal 66.9% 68.0% 67.6% 67.8% 71.3% R24.10.2 Cow Fecal 94.8% 61.1% 75.0% 99.9% 81.3% R24.10.7 Cow Fecal 99.5% 99.2% 98.8% 99.5% 97.7% R24.11.5 Cow Fecal 100.0% 100.0% 100.0% 99.9% 99.9% R24.11.6 Cow Fecal 99.7% 99.9% 99.7% 99.5% 99.3% R24.12.9 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R24.13.1 Cow Fecal 86.1% 93.7% 100.0% 91.9% 94.0% R24.13.4 Cow Fecal 75.8% 77.8% 76.2% 82.7% 85.9% R24.13.6 Cow Fecal 99.7% 100.0% 99.7% 99.9% 99.2% R24.13.7 Cow Fecal 99.8% 99.9% 99.9% 99.9% 99.7% R24.13.8 Cow Fecal 90.6% 96.9% 94.4% 91.5% 94.2% R24.13.9 Cow Fecal 98.0% 97.9% 97.9% 99.4% 98.9% R24.14.3 Cow Fecal 95.6% 93.3% 90.3% 90.6% 94.8% R24.14.7 Cow Fecal 95.8% 96.0% 95.5% 96.0% 95.8% R24.14.8 Cow Fecal 99.0% 99.6% 99.2% 99.7% 95.4% R24.14.9 Cow Fecal 100.0% 100.0% 100.0% 99.8% 99.8% R24.15.1 Cow Fecal 25.9% 0.0% 51.3% 89.5% 45.2% R24.15.10 Cow Fecal 93.4% 97.0% 99.5% 97.8% 96.0% R24.15.2 Cow Fecal 100.0% 99.6% 99.9% 99.9% 99.4% R24.15.4 Cow Fecal 89.7% 72.1% 91.0% 76.4% 99.4% R24.15.6 Cow Fecal 99.7% 99.9% 99.6% 99.6% 98.2% R24.15.7 Cow Fecal 0.0% 0.0% 0.0% 0.0% 0.0% R24.15.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R24.16.3 Cow Fecal 82.1% 13.6% 18.2% 43.3% 41.9% R24.16.6 Cow Fecal 99.3% 99.5% 6.7% 99.5% 71.1% R24.16.7 Cow Fecal 100.0% 100.0% 93.6% 100.0% 92.2% R24.17.1 Cow Fecal 99.9% 99.9% 100.0% 99.9% 99.9% R24.17.2 Cow Fecal 87.4% 97.2% 89.4% 0.0% 91.7% R24.17.4 Cow Fecal 100.0% 100.0% 93.1% 100.0% 99.9% R24.17.6 Cow Fecal 99.8% 99.9% 99.6% 99.8% 99.2% R24.17.7 Cow Fecal 86.5% 88.7% 77.0% 93.1% 84.9% R24.18.5 Cow Fecal 100.0% 100.0% 100.0% 100.0% 99.9% R24.18.9 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R24.19.1 Cow Fecal 99.3% 99.0% 0.0% 99.0% 17.1% R24.19.2 Cow Fecal 99.2% 99.0% 98.8% 99.0% 99.0% R24.19.3 Cow Fecal 99.8% 99.7% 99.7% 99.8% 99.5% R24.19.7 Cow Fecal 100.0% 99.8% 86.4% 99.8% 83.8% R24.2.2 Cow Fecal 99.4% 99.9% 99.7% 99.7% 99.7% R24.2.3 Cow Fecal 99.9% 100.0% 100.0% 100.0% 100.0% R24.2.7 Cow Fecal 99.6% 99.6% 99.6% 99.6% 99.0% R24.21.2 Cow Fecal 83.4% 36.2% 60.2% 91.5% 59.2% R24.21.6 Cow Fecal 100.0% 93.9% 80.3% 100.0% 97.7% R24.22.2 Cow Fecal 100.0% 100.0% 100.0% 99.8% 100.0% R24.22.4 Cow Fecal 100.0% 100.0% 100.0% 100.0% 99.7% R24.22.5 Cow Fecal 98.8% 99.0% 69.2% 98.8% 72.8% R24.23.2 Cow Fecal 99.0% 100.0% 97.8% 100.0% 99.6% R24.23.8 Cow Fecal 99.5% 99.9% 99.7% 98.7% 99.7% R24.23.9 Cow Fecal 99.9% 99.5% 99.6% 99.8% 99.3% R24.25.1 Cow Fecal 99.8% 99.5% 99.2% 99.6% 96.7% R24.27.10 Cow Fecal 99.9% 100.0% 99.8% 99.9% 99.6% R24.27.5 Cow Fecal 99.9% 99.6% 99.8% 99.9% 99.6% R24.27.7 Cow Fecal 99.7% 99.8% 100.0% 99.6% 99.8% R24.27.9 Cow Fecal 100.0% 99.8% 99.9% 99.8% 99.8% R24.28.10 Cow Fecal 90.2% 100.0% 100.0% 98.0% 99.0% R24.28.6 Cow Fecal 100.0% 100.0% 100.0% 100.0% 99.1% R24.28.8 Cow Fecal 100.0% 100.0% 99.9% 99.8% 99.0% R24.3.1 Cow Fecal 98.6% 99.3% 98.9% 99.2% 99.0% R24.3.5 Cow Fecal 99.7% 100.0% 99.9% 99.8% 99.7% R24.3.6 Cow Fecal 99.4% 100.0% 93.3% 99.4% 88.4% R24.31.1 Cow Fecal 100.0% 100.0% 100.0% 100.0% 99.6% R24.31.9 Cow Fecal 88.7% 88.7% 71.1% 75.8% 71.7% R24.32.8 Cow Fecal 98.7% 98.2% 96.8% 97.3% 93.0% R24.33.1 Cow Fecal 99.7% 99.9% 99.9% 99.8% 99.8% R24.34.5 Cow Fecal 97.6% 94.1% 0.0% 95.9% 77.2% R24.35.2 Cow Fecal 99.9% 100.0% 100.0% 100.0% 99.9% R24.36.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R24.37.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R24.38.3 Cow Fecal 90.3% 92.8% 0.0% 92.8% 29.2% R24.39.1 Cow Fecal 72.3% 0.0% 0.0% 0.0% 0.0% R24.39.10 Cow Fecal 100.2% 100.2% 99.0% 100.2% 99.0% R24.39.3 Cow Fecal 99.6% 100.0% 100.0% 99.8% 99.6% R24.39.5 Cow Fecal 83.1% 0.0% 100.0% 17.4% 99.9% R24.39.7 Cow Fecal 52.1% 52.4% 57.5% 59.9% 53.4% R24.4.10 Cow Fecal 100.0% 98.8% 96.9% 98.9% 99.5% R24.40.10 Cow Fecal 99.4% 56.7% 55.1% 100.0% 93.7% R24.40.5 Cow Fecal 100.0% 99.8% 100.0% 100.0% 99.8% R24.41.4 Cow Fecal 99.9% 100.0% 99.9% 100.0% 99.8% R24.42.1 Cow Fecal 100.0% 99.9% 99.5% 99.9% 98.9% R24.42.5 Cow Fecal 100.0% 99.9% 24.3% 100.0% 98.8% R24.44.1 Cow Fecal 99.9% 99.9% 99.5% 99.9% 100.0% R24.44.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R24.47.5 Cow Fecal 99.9% 99.9% 92.8% 99.9% 99.7% R24.47.8 Cow Fecal 99.6% 99.2% 99.5% 99.5% 97.7% R24.48.1 Cow Fecal 99.0% 98.6% 99.0% 98.6% 98.8% R24.5.1 Cow Fecal 99.8% 100.0% 100.0% 99.6% 99.6% R24.5.8 Cow Fecal 99.5% 99.5% 100.0% 99.5% 98.0% R24.50.6 Cow Fecal 98.7% 91.4% 75.3% 99.0% 88.5% R24.51.5 Cow Fecal 99.6% 99.4% 99.3% 99.4% 98.8% R24.52.10 Cow Fecal 98.4% 100.0% 97.1% 100.0% 100.0% R24.52.4 Cow Fecal 99.4% 99.1% 42.8% 99.4% 94.7% R24.52.6 Cow Fecal 60.5% 47.9% 48.9% 58.5% 55.7% R24.52.7 Cow Fecal 83.8% 84.3% 81.3% 77.9% 81.0% R24.52.9 Cow Fecal 97.1% 99.9% 100.0% 99.9% 99.9% R24.53.1 Cow Fecal 99.7% 99.6% 99.3% 99.8% 84.6% R24.54.10 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R24.55.1 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R24.55.2 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R24.59.2 Cow Fecal 100.0% 96.7% 45.3% 100.0% 83.8% R24.59.6 Cow Fecal 99.5% 47.3% 5.5% 100.0% 60.8% R24.6.3 Cow Fecal 99.6% 99.6% 99.4% 99.8% 96.7% R24.6.5 Cow Fecal 100.0% 15.9% 0.0% 100.0% 15.5% R24.6.9 Cow Fecal 100.0% 100.0% 100.0% 100.0% 99.8% R24.64.2 Cow Fecal 99.8% 99.7% 99.6% 99.7% 97.8% R24.65.7 Cow Fecal 100.0% 86.2% 96.4% 100.0% 100.0% R24.65.9 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R24.66.3 Cow Fecal 100.0% 99.6% 100.0% 85.9% 100.0% R24.66.4 Cow Fecal 52.4% 54.2% 49.8% 53.9% 54.9% R24.66.5 Cow Fecal 99.6% 99.4% 81.1% 99.6% 99.5% R24.66.9 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R24.67.1 Cow Fecal 0.0% 0.0% 0.0% 0.0% 0.0% R24.67.10 Cow Fecal 80.2% 27.6% 0.0% 15.6% 64.5% R24.7.10 Cow Fecal 75.8% 72.0% 77.2% 73.2% 85.5% R24.7.9 Cow Fecal 0.0% 8.4% 0.0% 0.0% 0.0% R24.70.3 Cow Fecal 97.6% 98.5% 85.7% 92.0% 96.7% R24.71.5 Cow Fecal 99.8% 100.0% 100.0% 99.4% 99.4% R24.71.6 Cow Fecal 99.8% 99.6% 99.5% 99.9% 99.0% R24.72.3 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R24.72.4 Cow Fecal 99.9% 100.0% 100.0% 99.9% 100.0% R24.72.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 99.9% R24.73.1 Cow Fecal 100.0% 82.8% 99.9% 99.9% 99.9% R24.73.2 Cow Fecal 99.7% 99.7% 100.0% 99.1% 99.9% R24.73.3 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R24.73.6 Cow Fecal 99.7% 99.9% 99.7% 99.6% 99.7% R24.73.7 Cow Fecal 25.0% 10.1% 16.2% 22.1% 24.3% R24.73.8 Cow Fecal 99.4% 99.8% 99.6% 99.6% 99.6% R24.73.9 Cow Fecal 100.0% 100.0% 100.0% 99.4% 100.0% R24.74.10 Cow Fecal 98.8% 99.1% 83.5% 99.0% 97.7% R24.74.5 Cow Fecal 92.8% 65.4% 72.1% 75.8% 82.9% R24.75.8 Cow Fecal 78.7% 88.0% 70.8% 73.9% 66.1% R24.76.3 Cow Fecal 99.7% 99.9% 100.0% 99.7% 99.9% R24.78.8 Cow Fecal 99.3% 99.3% 84.6% 99.3% 99.3% R24.80.10 Cow Fecal 100.0% 100.0% 99.9% 98.9% 99.9% R24.81.3 Cow Fecal 99.6% 99.8% 98.0% 99.7% 99.3% R24.81.4 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R24.81.6 Cow Fecal 100.0% 100.0% 100.0% 100.0% 99.9% R24.81.9 Cow Fecal 99.7% 99.1% 93.1% 99.5% 99.3% R24.82.10 Cow Fecal 99.5% 99.3% 84.7% 99.7% 98.8% R24.82.5 Cow Fecal 99.9% 99.7% 36.8% 99.8% 99.0% R24.82.7 Cow Fecal 98.2% 98.7% 59.4% 98.6% 96.7% R24.82.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R24.9.10 Cow Fecal 99.4% 99.9% 99.1% 99.3% 98.2% R25.10.3 Calf Fecal 99.9% 100.0% 99.2% 99.9% 99.9% R25.10.5 Calf Fecal 99.8% 100.0% 99.6% 99.9% 99.8% R25.10.6 Calf Fecal 99.9% 100.0% 100.0% 100.0% 100.0% R25.10.7 Calf Fecal 100.0% 100.0% 99.5% 100.0% 99.9% R25.14.4 Calf Fecal 96.9% 76.8% 35.5% 89.5% 80.6% R25.16.1 Cow Fecal 84.6% 79.0% 70.8% 83.9% 69.0% R25.16.8 Cow Fecal 99.5% 100.0% 99.8% 100.0% 100.0% R25.17.2 Cow Fecal 99.8% 99.8% 99.6% 99.8% 99.6% R25.18.10 Cow Fecal 99.8% 100.0% 99.3% 98.9% 99.2% R25.18.3 Cow Fecal 99.9% 100.0% 99.3% 98.7% 98.7% R25.18.6 Cow Fecal 99.9% 100.0% 100.0% 100.0% 100.0% R25.20.1 Cow Fecal 99.6% 100.0% 98.9% 98.5% 99.4% R25.20.3 Cow Fecal 99.2% 100.0% 99.2% 100.0% 99.7% R25.20.5 Cow Fecal 98.3% 99.8% 99.1% 99.7% 99.0% R25.21.4 Cow Fecal 99.9% 100.0% 99.6% 100.0% 99.6% R25.21.7 Cow Fecal 0.0% 0.0% 0.0% 0.0% 0.0% R25.22.2 Cow Fecal 100.0% 94.4% 71.1% 99.1% 85.3% R25.22.8 Cow Fecal 99.9% 99.9% 99.9% 100.0% 99.9% R25.23.9 Cow Fecal 99.9% 100.0% 99.6% 100.0% 98.6% R25.24.8 Cow Fecal 85.4% 45.5% 15.9% 35.8% 52.4% R25.25.2 Cow Fecal 100.0% 99.9% 99.2% 98.8% 100.0% R25.26.1 Cow Fecal 99.5% 99.1% 98.7% 97.3% 97.9% R25.26.10 Cow Fecal 100.0% 98.3% 98.8% 97.4% 98.0% R25.26.7 Cow Fecal 99.7% 99.7% 99.3% 98.5% 99.7% R25.26.9 Cow Fecal 100.0% 99.9% 99.8% 98.6% 99.8% R25.27.7 Cow Fecal 99.9% 99.4% 89.5% 97.8% 94.2% R25.28.1 Cow Fecal 99.8% 100.0% 100.0% 98.8% 99.6% R25.28.5 Cow Fecal 80.0% 75.3% 80.4% 51.4% 39.7% R25.29.1 Cow Fecal 99.8% 99.5% 98.4% 97.8% 98.4% R25.29.2 Cow Fecal 100.0% 99.8% 98.5% 95.5% 98.2% R25.31.10 Cow Fecal 99.9% 99.9% 99.0% 98.2% 99.6% R25.31.2 Cow Fecal 98.8% 88.9% 87.2% 79.8% 79.0% R25.31.4 Cow Fecal 99.9% 99.7% 99.0% 99.1% 99.6% R25.31.5 Cow Fecal 100.0% 100.0% 99.7% 98.6% 99.8% R25.31.8 Cow Fecal 99.9% 99.4% 99.4% 97.7% 98.2% R25.32.10 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R25.32.6 Cow Fecal 99.8% 99.8% 90.1% 99.4% 99.7% R25.32.8 Cow Fecal 100.0% 99.9% 99.9% 100.0% 99.9% R25.32.9 Cow Fecal 99.8% 99.8% 99.8% 98.4% 99.5% R25.33.4 Cow Fecal 91.9% 98.9% 4.2% 89.1% 95.4% R25.33.9 Cow Fecal 99.9% 100.0% 100.0% 99.8% 99.8% R25.34.7 Cow Fecal 100.0% 99.8% 98.7% 98.4% 99.8% R25.34.9 Cow Fecal 99.8% 98.8% 96.5% 98.1% 99.8% R25.35.2 Cow Fecal 99.8% 99.9% 100.0% 99.3% 99.8% R25.37.10 Cow Fecal 100.0% 100.0% 100.0% 99.7% 100.0% R25.37.2 Cow Fecal 99.8% 99.8% 98.5% 99.7% 99.9% R25.37.3 Cow Fecal 100.0% 100.0% 99.7% 99.7% 99.9% R25.37.8 Cow Fecal 100.0% 100.0% 99.9% 99.7% 99.9% R25.38.3 Cow Fecal 99.3% 99.8% 100.0% 99.1% 99.6% R25.39.3 Cow Fecal 100.0% 99.9% 100.0% 100.0% 99.9% R25.39.7 Cow Fecal 99.9% 99.5% 99.9% 100.0% 99.8% R25.39.9 Cow Fecal 99.9% 99.8% 96.9% 99.9% 99.9% R25.40.5 Cow Fecal 100.0% 99.8% 98.8% 98.9% 99.8% R25.40.6 Cow Fecal 99.9% 96.0% 84.9% 94.8% 99.2% R25.40.7 Cow Fecal 99.9% 100.0% 100.0% 99.6% 100.0% R25.41.2 Cow Fecal 99.0% 98.6% 89.6% 97.5% 95.1% R25.41.3 Cow Fecal 99.5% 92.7% 88.3% 98.8% 98.5% R25.41.5 Cow Fecal 97.2% 98.8% 99.2% 98.1% 99.8% R25.41.6 Cow Fecal 99.2% 99.2% 97.8% 99.2% 98.9% R25.42.6 Cow Fecal 99.3% 99.2% 99.2% 98.0% 98.6% R25.43.2 Cow Fecal 98.2% 98.6% 15.1% 98.7% 78.0% R25.43.4 Cow Fecal 98.2% 99.1% 81.1% 97.9% 97.6% R25.43.6 Cow Fecal 99.7% 99.3% 99.4% 97.7% 98.8% R25.43.7 Cow Fecal 100.0% 99.7% 99.0% 97.8% 99.9% R25.43.9 Cow Fecal 99.9% 99.8% 98.5% 98.0% 99.5% R25.44.5 Cow Fecal 96.7% 93.4% 96.7% 89.4% 96.0% R25.44.6 Cow Fecal 91.5% 76.1% 0.0% 86.4% 87.4% R25.45.2 Cow Fecal 100.0% 100.0% 99.9% 100.0% 99.9% R25.45.6 Cow Fecal 99.8% 99.4% 95.7% 94.1% 98.2% R25.46.3 Cow Fecal 87.3% 40.8% 0.0% 82.7% 61.4% R25.47.9 Cow Fecal 92.2% 83.3% 0.0% 87.5% 91.8% R25.48.8 Cow Fecal 0.0% 0.0% 0.0% 0.0% 0.0% R25.49.4 Cow Fecal 99.4% 98.8% 98.1% 97.0% 98.1% R25.49.5 Cow Fecal 99.5% 99.4% 98.8% 95.4% 98.2% R25.51.8 Cow Fecal 94.2% 85.5% 0.0% 84.5% 89.4% R25.52.2 Cow Fecal 98.7% 89.3% 72.7% 92.3% 83.4% R25.52.9 Cow Fecal 99.1% 0.0% 4.4% 81.1% 66.8% R25.53.3 Cow Fecal 95.1% 79.8% 14.8% 89.2% 86.9% R25.56.3 Cow Fecal 17.6% 0.0% 0.0% 0.0% 0.0% R25.57.5 Cow Fecal 73.1% 87.3% 63.8% 94.3% 66.0% R25.58.1 Cow Fecal 0.0% 0.0% 0.0% 0.0% 0.0% R25.63.1 Cow Fecal 4.7% 14.0% 0.0% 10.0% 0.0% R25.63.3 Cow Fecal 99.0% 40.0% 0.0% 93.1% 99.0% R25.67.5 Cow Fecal 63.9% 75.8% 91.3% 63.9% 76.7% R25.70.10 Cow Fecal 92.4% 79.1% 51.2% 94.6% 89.2% R25.70.4 Cow Fecal 57.3% 15.8% 39.6% 87.0% 50.6% R25.70.8 Cow Fecal 65.8% 61.7% 87.2% 72.5% 63.1% R25.72.2 Cow Fecal 99.0% 99.9% 100.0% 99.0% 100.0% R25.73.2 Cow Fecal 26.1% 0.0% 52.2% 0.0% 69.6% R25.73.3 Cow Fecal 98.7% 98.9% 99.9% 99.7% 98.9% R25.76.2 Cow Fecal 98.2% 99.9% 99.8% 99.0% 99.3% R25.76.5 Cow Fecal 99.9% 100.0% 100.0% 98.0% 100.0% R25.79.10 Cow Fecal 97.2% 96.6% 67.2% 94.6% 93.7% R25.79.9 Cow Fecal 95.7% 84.4% 35.9% 94.0% 86.6% R25.80.5 Cow Fecal 50.7% 42.2% 28.8% 46.6% 39.9% R25.82.3 Cow Fecal 76.8% 69.0% 71.4% 84.1% 74.5% R25.83.5 Cow Fecal 38.0% 97.0% 47.3% 65.3% 87.0% R25.85.1 Cow Fecal 78.2% 58.4% 48.8% 69.9% 71.6% R25.86.3 Cow Fecal 99.9% 61.5% 48.1% 75.2% 97.8% R25.86.6 Cow Fecal 56.9% 40.3% 25.3% 65.9% 66.9% R25.86.8 Cow Fecal 78.8% 62.0% 59.8% 78.9% 63.5% R26.1.4 Calf Fecal 51.3% 54.4% 90.2% 42.1% 67.4% R26.1.5 Calf Fecal 78.4% 56.5% 73.9% 77.3% 80.9% R26.10.3 Calf Fecal 99.0% 59.8% 17.1% 78.7% 49.8% R26.11.10 Cow Fecal 55.0% 39.3% 11.2% 45.2% 37.4% R26.11.9 Cow Fecal 38.9% 54.0% 0.0% 66.1% 49.5% R26.13.3 Cow Fecal 99.7% 47.2% 17.7% 99.0% 59.6% R26.13.6 Cow Fecal 100.0% 100.0% 99.8% 99.5% 99.8% R26.14.2 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R26.15.2 Cow Fecal 98.0% 100.0% 95.9% 99.0% 100.0% R26.15.3 Cow Fecal 41.6% 55.0% 45.0% 57.2% 52.3% R26.15.7 Cow Fecal 93.8% 97.9% 95.6% 95.6% 48.0% R26.16.8 Cow Fecal 95.8% 69.1% 60.2% 70.9% 86.1% R26.18.5 Cow Fecal 95.5% 76.7% 63.0% 77.7% 88.9% R26.19.4 Cow Fecal 77.9% 90.6% 72.3% 57.2% 80.5% R26.19.6 Cow Fecal 100.0% 100.0% 94.2% 89.7% 96.8% R26.2.4 Calf Fecal 100.0% 100.0% 100.0% 0.0% 100.0% R26.20.3 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R26.20.4 Cow Fecal 100.0% 33.3% 49.2% 36.4% 100.0% R26.20.6 Cow Fecal 11.4% 24.3% 9.5% 0.0% 0.0% R26.20.7 Cow Fecal 100.0% 100.0% 100.0% 97.2% 96.4% R26.21.10 Cow Fecal 100.0% 100.0% 99.1% 99.6% 100.0% R26.21.4 Cow Fecal 90.3% 80.8% 75.9% 84.6% 88.0% R26.21.7 Cow Fecal 100.0% 94.6% 95.7% 96.6% 99.3% R26.21.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R26.23.10 Cow Fecal 92.9% 97.3% 96.7% 98.8% 98.0% R26.23.2 Cow Fecal 99.7% 99.7% 98.8% 100.0% 98.9% R26.23.3 Cow Fecal 100.0% 99.9% 96.2% 97.5% 89.3% R26.23.6 Cow Fecal 100.0% 100.0% 98.5% 97.7% 98.5% R26.23.7 Cow Fecal 68.3% 78.7% 71.7% 85.0% 85.7% R26.23.8 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R26.23.9 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R26.24.9 Cow Fecal 100.0% 99.8% 98.4% 97.4% 98.9% R26.26.2 Cow Fecal 100.0% 99.8% 97.6% 97.9% 99.4% R26.26.4 Cow Fecal 0.0% 13.2% 0.0% 0.0% 0.0% R26.27.8 Cow Fecal 99.8% 98.1% 97.2% 95.7% 97.6% R26.28.6 Cow Fecal 99.9% 99.6% 99.6% 97.9% 98.7% R26.28.8 Cow Fecal 96.5% 98.1% 97.9% 95.7% 97.3% R26.3.5 Calf Fecal 27.3% 0.0% 38.9% 63.2% 99.4% R26.30.10 Cow Fecal 0.0% 33.8% 89.2% 0.0% 0.0% R26.30.2 Cow Fecal 99.7% 97.4% 98.0% 94.0% 96.0% R26.31.3 Cow Fecal 92.6% 90.7% 99.3% 92.5% 98.6% R26.31.8 Cow Fecal 100.0% 99.5% 99.3% 97.9% 98.9% R26.32.1 Cow Fecal 99.5% 98.6% 95.5% 94.8% 97.1% R26.33.4 Cow Fecal 99.9% 99.3% 97.2% 97.0% 98.7% R26.34.5 Cow Fecal 100.0% 98.3% 97.1% 96.3% 98.0% R26.35.5 Cow Fecal 99.6% 99.5% 96.8% 96.7% 98.5% R26.38.1 Cow Fecal 99.3% 98.6% 96.1% 96.1% 97.2% R26.38.10 Cow Fecal 99.9% 97.7% 97.4% 96.5% 97.5% R26.39.6 Cow Fecal 99.9% 98.4% 96.3% 96.6% 97.5% R26.41.10 Cow Fecal 100.0% 93.1% 2.0% 87.9% 92.7% R26.41.2 Cow Fecal 98.9% 97.8% 87.1% 89.2% 93.5% R26.43.6 Cow Fecal 0.0% 0.0% 0.0% 0.0% 0.0% R26.44.7 Cow Fecal 100.1% 100.0% 98.9% 99.5% 100.1% R26.47.4 Cow Fecal 100.0% 99.1% 94.6% 95.5% 100.0% R26.5.1 Calf Fecal 100.0% 100.0% 100.0% 99.2% 100.0% R26.5.2 Calf Fecal 72.8% 86.6% 66.1% 98.4% 79.2% R26.5.5 Calf Fecal 91.1% 100.0% 98.5% 98.4% 99.2% R26.5.9 Calf Fecal 65.7% 74.1% 50.3% 93.0% 94.1% R26.51.1 Cow Fecal 100.0% 100.0% 99.2% 99.3% 100.0% R26.52.3 Cow Fecal 91.1% 89.0% 7.9% 84.7% 87.6% R26.52.5 Cow Fecal 76.9% 75.1% 77.2% 78.3% 81.0% R26.52.6 Cow Fecal 85.6% 84.6% 83.6% 81.3% 78.7% R26.53.10 Cow Fecal 100.0% 100.0% 99.3% 98.5% 100.0% R26.54.4 Cow Fecal 100.0% 100.0% 99.3% 99.4% 100.0% R26.54.8 Cow Fecal 100.0% 100.0% 100.0% 99.0% 99.7% R26.55.4 Cow Fecal 93.7% 76.3% 9.0% 95.2% 39.0% R26.55.5 Cow Fecal 100.0% 99.3% 100.0% 98.6% 100.0% R26.56.1 Cow Fecal 100.0% 99.8% 99.8% 98.0% 100.0% R26.56.6 Cow Fecal 100.0% 99.9% 99.1% 98.9% 100.0% R26.57.6 Cow Fecal 99.6% 98.7% 97.9% 98.6% 98.9% R26.57.9 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R26.59.1 Cow Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R26.59.10 Cow Fecal 100.0% 99.9% 99.9% 99.5% 100.0% R26.59.6 Cow Fecal 99.9% 100.0% 100.0% 100.0% 100.0% R26.59.7 Cow Fecal 100.0% 100.0% 99.3% 98.7% 100.0% R26.59.8 Cow Fecal 99.7% 99.6% 99.6% 99.6% 99.6% R26.60.7 Cow Fecal 99.9% 99.3% 99.4% 99.2% 99.5% R26.8.4 Calf Fecal 100.0% 94.4% 98.7% 87.3% 98.8% R26.8.9 Calf Fecal 99.9% 94.5% 63.4% 29.2% 96.8% R26.9.10 Calf Fecal 97.4% 50.7% 0.0% 88.4% 46.4% R26.9.3 Calf Fecal 63.4% 44.5% 99.9% 57.3% 68.6% R26.9.6 Calf Fecal 89.8% 48.9% 99.6% 99.6% 100.0% R26.9.7 Calf Fecal 0.0% 47.4% 52.1% 0.0% 41.6% R26.9.8 Calf Fecal 92.2% 99.7% 98.1% 97.7% 93.6% R1.10.4 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.10.5 Calf Fecal 100.0% 100.0% 100.0% 100.0% 99.9% R1.11.3 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.11.4 Calf Fecal 100.0% 100.0% 99.9% 100.0% 100.0% R1.12.10 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.12.5 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.12.8 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.13.1 Calf Fecal 100.0% 0.6% 98.5% 27.9% 100.0% R1.14.3 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.15.1 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.15.10 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.15.2 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.15.4 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.15.7 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.15.9 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.16.1 Calf Fecal 100.0% 99.8% 100.0% 100.0% 100.0% R1.16.6 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.17.2 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.18.1 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.18.10 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.18.2 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.18.4 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.18.6 Calf Fecal 0.0% 0.0% 0.0% 0.0% 0.0% R1.18.7 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.18.9 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.19.3 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.21.2 Calf Fecal 100.0% 99.8% 100.0% 100.0% 100.0% R1.21.4 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.22.2 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.22.4 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.22.6 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.22.9 Calf Fecal 99.3% 99.7% 99.6% 99.5% 99.6% R1.23.5 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.23.8 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.23.9 Calf Fecal 99.9% 100.0% 100.0% 100.0% 100.0% R1.24.1 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.24.8 Calf Fecal 100.0% 100.0% 100.0% 99.9% 100.0% R1.25.1 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.25.2 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.25.4 Calf Fecal 100.0% 100.0% 100.0% 99.8% 100.0% R1.25.5 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.25.7 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.26.4 Calf Fecal 100.0% 12.0% 98.0% 0.2% 100.0% R1.27.6 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.27.9 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.28.4 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.34.3 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.34.8 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.35.7 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.46.2 Calf Fecal 100.0% 100.0% 100.0% 99.8% 100.0% R1.46.4 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.9.4 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0% R1.9.9 Calf Fecal 100.0% 100.0% 100.0% 100.0% 100.0%

TABLE 12 Bacteriocin assay results displaying each isolate tested from Texas. Inhibition was calculated based on the percent of growth for each treatment well compared to the positive control. Isolate Sample Bacillus Strains 16S ID ID Type Farm 747 1104 1541 1781 1999 2018 C. beijerinckii group R3.9.4 Cow Fecal Farm BS 98.7% 98.8% 98.1% 71.2% 76.8% 99.7% C. beijerinckii group S37.11.9 Ground Corn Farm BS 96.9% 45.7% 62.0% 39.3% 36.9% 66.2% C. beijerinckii group R3.60.2 Cow Fecal Farm BS 99.0% 99.0% 98.5% 99.1% 79.9% 98.7% C. beijerinckii group R5.15.1 Cow Fecal Farm ALJ 98.6% 79.9% 97.8% 49.3% 52.4% 80.0% C. beijerinckii group R5.42.1 Cow Fecal Farm ALJ 75.6% 53.9% 96.5% 51.2% 70.6% 79.9% C. beijerinckii group R5.41.1 Cow Fecal Farm ALJ 97.2% 64.2% 94.5% 26.0% 24.6% 42.5% C. bifermentans group S37.12.5 Gluten Feed Farm BS 96.0% 70.0% 68.5% 69.3% 74.8% 69.5% C. butyricum R1.26.7 Calf Fecal Farm DC 90.7% 26.9% 35.3% 52.9% 40.9% 72.8% C. butyricum R3.5.7 Cow Fecal Farm BS 99.1% 99.3% 98.7% 79.8% 79.3% 99.2% C. butyricum R3.4.5 Cow Fecal Farm BS 98.5% 98.2% 97.5% 97.7% 97.5% 97.5% C. butyricum S35.1.1 Texture Feed #1 Farm DC 76.8% 51.8% 41.4% 43.8% 41.2% 76.3% C. paraputrificum R1.10.7 Calf Fecal Farm DC 97.1% 97.6% 96.7% 90.4% 95.3% 97.1% C. paraputrificum R1.19.10 Calf Fecal Farm DC 94.0% 85.4% 61.2% 50.4% 81.0% 48.6% Clostrdium R1.12.7 Calf Fecal Farm DC 96.9% 33.3% 96.8% 97.0% Not Tested 97.0% perfringens Clostrdium R1.12.4 Calf Fecal Farm DC 96.3% 22.2% 96.2% 96.1% Not Tested 96.7% perfringens Clostrdium tertium R1.3.10 Calf Fecal Farm DC 82.5% 5.6% 22.3% 33.5% Not Tested 89.8% Clostrdium tertium R1.3.9 Calf Fecal Farm DC 87.4% 35.6% 47.0% 52.8% Not Tested 93.8% Clostrdium tertium R1.17.3 Calf Fecal Farm DC 85.2% 2.9% 19.2% 32.0% Not Tested 92.5% Clostrdium tertium R1.20.10 Calf Fecal Farm DC 85.3% 0.0% 33.3% 35.7% Not Tested 92.0% Clostrdium tertium R1.20.9 Calf Fecal Farm DC 82.9% 1.0% 21.8% 31.7% Not Tested 92.0% Clostrdium tertium R1.19.9 Calf Fecal Farm DC 84.1% 27.7% 39.7% 58.6% Not Tested 89.3% Clostrdium tertium R1.36.7 Calf Fecal Farm DC 85.2% 9.8% 25.9% 32.7% Not Tested 96.2% Clostrdium tertium R1.32.5 Calf Fecal Farm DC 96.9% 14.2% 77.4% 60.3% Not Tested 96.6% Clostrdium tertium R1.39.4 Calf Fecal Farm DC 97.2% 6.1% 60.5% 69.4% Not Tested 96.8% Clostrdium tertium R1.33.8 Calf Fecal Farm DC 71.4% 31.8% 24.2% 47.6% Not Tested 83.0% Clostrdium tertium R1.32.6 Calf Fecal Farm DC 89.2% 0.0% 10.0% 25.7% Not Tested 95.9% Clostrdium tertium R1.33.9 Calf Fecal Farm DC 70.7% 27.8% 17.4% 45.3% Not Tested 60.0% Clostrdium tertium R1.41.8 Calf Fecal Farm DC 80.7% 0.0% 25.8% 29.2% Not Tested 91.0% Clostrdium tertium R1.46.7 Calf Fecal Farm DC 96.2% 45.5% 64.7% 71.9% Not Tested 96.5% Clostridium R5.35.6 Cow Fecal Farm ALJ 37.3% 30.7% 39.1% 38.6% Not Tested 37.5% algidixylanolyticum Clostridium R3.13.1 Cow Fecal Farm BS 13.6% 21.4% 9.6% 15.2% Not Tested 12.4% algidixyllanolyticum Clostridium R3.2.3 Cow Fecal Farm BS 42.8% 34.3% 28.9% 37.7% Not Tested 27.8% algidixyllanolyticum Clostridium R3.36.2 Cow Fecal Farm BS 25.6% 21.8% 25.6% 24.8% Not Tested 25.7% algidixyllanolyticum Clostridium R3.37.2 Cow Fecal Farm BS 23.0% 11.8% 22.1% 20.4% Not Tested 19.2% algidixyllanolyticum Clostridium R3.47.7 Cow Fecal Farm BS 15.8% 0.0% 10.2% 11.6% Not Tested 11.8% algidixyllanolyticum Clostridium R3.50.1 Cow Fecal Farm BS 5.5% 0.0% 4.4% 4.5% Not Tested 5.6% algidixyllanolyticum Clostridium R3.53.4 Cow Fecal Farm BS 0.0% 0.0% 0.0% 0.0% Not Tested 0.0% algidixyllanolyticum Clostridium R3.55.7 Cow Fecal Farm BS 6.6% 4.1% 5.2% 7.3% Not Tested 7.7% algidixyllanolyticum Clostridium R5.59.10 Cow Fecal Farm ALJ 95.6% 71.1% 90.4% 90.9% Not Tested 94.3% argentinense Clostridium R3.21.4 Cow Fecal Farm BS 26.4% 26.9% 26.3% 27.5% Not Tested 26.3% beijerinkckii group Clostridium R3.17.2 Cow Fecal Farm BS 11.6% 10.1% 15.1% 10.3% Not Tested 16.8% beijerinkckii group Clostridium R3.16.2 Cow Fecal Farm BS 21.2% 24.8% 22.3% 23.2% Not Tested 23.1% beijerinkckii group Clostridium R3.38.3 Cow Fecal Farm BS 27.0% 26.4% 26.7% 25.7% Not Tested 30.4% beijerinkckii group Clostridium R3.33.1 Cow Fecal Farm BS 19.5% 18.5% 19.0% 18.7% Not Tested 17.2% beijerinkckii group Clostridium R3.45.5 Cow Fecal Farm BS 28.5% 28.0% 27.3% 27.5% Not Tested 27.6% beijerinkckii group Clostridium R3.52.2 Cow Fecal Farm BS 23.7% 21.4% 19.7% 19.1% Not Tested 18.7% beijerinkckii group Clostridium R3.43.3 Cow Fecal Farm BS 31.3% 29.7% 30.2% 30.9% Not Tested 31.2% beijerinkckii group Clostridium R3.59.4 Cow Fecal Farm BS 8.3% 26.7% 12.3% 14.0% Not Tested 8.3% beijerinkckii group Clostridium S37.11.3 Ground Corn Farm BS 24.1% 21.9% 25.5% 21.1% Not Tested 28.9% beijerinkckii group Clostridium S37.11.2 Ground Corn Farm BS 24.3% 22.8% 21.8% 20.0% Not Tested 20.3% beijerinkckii group Clostridium R5.1.1 Cow Fecal Farm ALJ 23.9% 23.6% 22.5% 21.0% Not Tested 20.5% beijerinkckii group Clostridium R5.12.4 Cow Fecal Farm ALJ 17.9% 17.6% 20.6% 19.7% Not Tested 39.8% beijerinkckii group Clostridium R5.23.10 Cow Fecal Farm ALJ 59.8% 71.7% 64.9% 68.1% Not Tested 65.9% beijerinkckii group Clostridium R5.24.3 Cow Fecal Farm ALJ 66.1% 77.3% 50.6% 79.3% Not Tested 51.8% beijerinkckii group Clostridium R5.19.1 Cow Fecal Farm ALJ 21.7% 22.3% 20.3% 21.9% Not Tested 22.3% beijerinkckii group Clostridium R5.14.10 Cow Fecal Farm ALJ 20.0% 22.1% 17.7% 17.8% Not Tested 18.6% beijerinkckii group Clostridium R5.21.1 Cow Fecal Farm ALJ 89.1% 7.0% 7.3% 86.7% Not Tested 7.5% beijerinkckii group Clostridium R5.38.3 Cow Fecal Farm ALJ 71.2% 0.0% 16.0% 1.3% Not Tested 40.9% beijerinkckii group Clostridium R5.27.9 Cow Fecal Farm ALJ 27.9% 26.9% 34.7% 27.6% Not Tested 26.5% beijerinkckii group Clostridium R5.28.1 Cow Fecal Farm ALJ 16.6% 17.3% 17.9% 17.0% Not Tested 21.1% beijerinkckii group Clostridium R5.50.1 Cow Fecal Farm ALJ 22.5% 23.7% 20.5% 23.2% Not Tested 18.8% beijerinkckii group Clostridium R5.59.1 Cow Fecal Farm ALJ 20.0% 23.2% 27.4% 21.5% Not Tested 21.9% beijerinkckii group Clostridium R5.41.1 Cow Fecal Farm ALJ 21.6% 24.1% 22.3% 19.8% Not Tested 20.6% beijerinkckii group Clostridium R3.31.4 Cow Fecal Farm BS 95.2% 90.4% 94.6% 94.6% Not Tested 94.9% bifermentans group Clostridium S37.11.4 Ground Corn Farm BS 97.3% 97.2% 96.5% 96.4% Not Tested 97.0% bifermentans group Clostridium R5.20.1 Cow Fecal Farm ALJ 97.1% 65.3% 96.3% 96.1% Not Tested 96.8% bifermentans group Clostridium R5.53.1 Cow Fecal Farm ALJ 92.2% 0.0% 89.9% 89.0% Not Tested 90.1% bifermentans group Clostridium R5.52.1 Cow Fecal Farm ALJ 96.9% 28.1% 95.9% 78.5% Not Tested 95.3% bifermentans group Clostridium R1.26.7 Farm DC 96.3% 0.0% 40.4% 0.0% Not Tested 91.4% butyricum Clostridium R1.46.8 Calf Fecal Farm DC 97.6% 97.7% 97.1% 97.1% Not Tested 97.2% butyricum Clostridium R1.21.3 Calf Fecal Farm DC 97.0% 0.0% 82.4% 0.0% Not Tested 97.2% cadaveris Clostridium R1.30.6 Calf Fecal Farm DC 96.7% 0.0% 91.2% 0.0% Not Tested 96.6% cadaveris Clostridium R1.37.1 Calf Fecal Farm DC 97.7% 0.4% 90.2% 18.0% Not Tested 97.7% cadaveris Clostridium R1.45.1 Calf Fecal Farm DC 97.6% 0.0% 89.9% 38.8% Not Tested 97.0% cadaveris Clostridium R1.45.2 Calf Fecal Farm DC 97.4% 0.0% 89.6% 48.2% Not Tested 96.6% cadaveris Clostridium R3.22.2 Cow Fecal Farm BS 95.5% 0.0% 83.3% 45.7% Not Tested 94.2% cadaveris Clostridium R3.26.9 Cow Fecal Farm BS 97.6% 0.0% 94.2% 55.2% Not Tested 95.8% cadaveris Clostridium R5.11.10 Cow Fecal Farm ALJ 88.5% 87.6% 86.8% 85.6% Not Tested 87.0% cadaveris Clostridium R5.26.3 Cow Fecal Farm ALJ 14.9% 10.5% 15.7% 14.5% Not Tested 13.6% celerecrescens Clostridium ghonii S37.11.1 Ground Corn Farm BS 24.5% 23.1% 23.0% 23.6% Not Tested 24.2% Clostridium ghonii S37.12.4 Gluten Feed Farm BS 93.5% 0.0% 0.0% 15.3% Not Tested 27.6% Clostridium ghonii S37.11.6 Ground Corn Farm BS 75.5% 0.0% 7.8% 74.8% Not Tested 76.2% Clostridium ghonii R5.17.1 Cow Fecal Farm ALJ 32.5% 0.0% 0.0% 0.0% Not Tested 0.0% Clostridium R1.11.9 Calf Fecal Farm DC 96.0% 53.3% 81.2% 86.3% Not Tested 96.1% paraputrificum Clostridium R1.17.1 Calf Fecal Farm DC 94.3% 17.9% 87.7% 79.8% Not Tested 91.8% paraputrificum Clostridium R1.19.10 Calf Fecal Farm DC 97.1% 56.9% 89.2% 91.8% Not Tested 96.1% paraputrificum Clostridium R1.31.4 Calf Fecal Farm DC 96.0% 29.9% 93.5% 89.8% Not Tested 96.0% paraputrificum Clostridium R1.40.3 Calf Fecal Farm DC 96.4% 50.9% 81.1% 85.5% Not Tested 96.0% paraputrificum Clostridium R1.44.3 Calf Fecal Farm DC 95.8% 38.1% 88.1% 73.6% Not Tested 97.0% paraputrificum Clostridium R1.43.8 Calf Fecal Farm DC 96.8% 35.3% 95.0% 92.4% Not Tested 97.0% paraputrificum Clostridium R3.14.3 Cow Fecal Farm BS 44.1% 49.8% 63.5% 71.0% Not Tested 70.0% sartagoforme Clostridium R3.29.4 Cow Fecal Farm BS 32.2% 23.0% 36.5% 34.2% Not Tested 34.1% sartagoforme Clostridium R3.46.1 Cow Fecal Farm BS 83.6% 0.0% 52.0% 43.3% Not Tested 64.5% sartagoforme Clostridium S37.11.5 Ground Corn Farm BS 96.0% 36.6% 77.4% 80.0% Not Tested 80.7% sartagoforme Clostridium S37.11.7 Ground Corn Farm BS 89.8% 30.8% 47.9% 70.0% Not Tested 79.0% sartagoforme Clostridium S37.1.1 Face Corn Farm BS 94.8% 0.0% 34.2% 29.3% Not Tested 84.0% sartagoforme Clostridium S37.11.8 Ground Corn Farm BS 94.1% 0.0% 33.9% 0.0% Not Tested 57.4% sartagoforme Clostridium R5.48.10 Cow Fecal Farm ALJ 92.4% 32.3% 74.8% 83.1% Not Tested 91.6% sartagoforme Clostridium R5.57.10 Cow Fecal Farm ALJ 87.6% 1.6% 42.2% 42.8% Not Tested 64.1% sartagoforme Clostridium sordellii S37.12.3 Gluten Feed Farm BS 97.2% 49.4% 96.0% 96.3% Not Tested 86.9% Clostridium R3.25.10 Cow Fecal Farm BS 74.9% 0.0% 8.1% 0.0% Not Tested 8.0% sporogenes Clostridium R5.47.3 Cow Fecal Farm ALJ 77.1% 57.9% 72.5% 49.8% Not Tested 75.3% subterminale Clostridium R5.56.1 Cow Fecal Farm ALJ 77.0% 0.0% 55.2% 48.2% Not Tested 59.8% subterminale Clostridium R5.45.2 Cow Fecal Farm ALJ 55.8% 46.9% 50.2% 46.5% Not Tested 52.2% sulfidigenes Clostridium R3.34.4 Cow Fecal Farm BS 90.1% 85.8% 84.8% 83.5% Not Tested 84.3% uliginosum

Example 7: Selection of Bacillus Strains to Inhibit Clostridium Perfringens and Non-Toxigenic Clostridia Isolated from Ruminant Fecal Samples. (Upper Midwest)

Introduction:

Clostridium is a genus of Gram-positive, spore-forming bacteria that are common residents of the gastrointestinal tract. A number of Clostridium species have been linked to enteric disease in ruminants including hemorrhagic bowel syndrome (HBS), a disease often correlated to elevated levels of Clostridium perfringens Type A. While most of the enteric diseases caused by clostridia are acute and occur sporadically in herds, in general, the prognosis is poor and the first sign of illness may be death. Based on recent results sub-acute enteric clostridia disease challenges may be a more wide spread issue than acute challenges. Due to a low success rate from treatment in acute disease challenges a more common, emphasis needs to be placed on prophylactic measures.

The purpose of this research was to characterize the distribution and diversity of clostridia in ruminants and ensure inhibition of these isolates using novel Bacillus strains as a method to control the clostridia populations.

Materials and Methods:

Fecal samples (248) from cows, heifers and calves gathered from 4 farms in the Upper Midwest region were diluted 1:10 with sterile peptone, heat shocked for 30 minutes at 60° C., enumerated in sterile peptone and pour plated on Tryptose Sulphite Cycloserine (TSC) agar with D-cycloserine (400 mg/L) to select for clostridia species. Agar plates were incubated at 37° C. anaerobically for 24 hours. If present, isolated sulphite-reducing colonies were counted for a total clostridia count (CFU/g) and representative isolates were picked into Reinforced clostridia Medium (RCM) (Oxoid, CM0149) and incubated anaerobically for 24 hours at 37° C. After 24 hours of incubation the cultures were transferred (10%) to Brain Heart Infusion (BHI) broth (BD, 211059) and incubated anaerobically for 24 hours at 37° C.

DNA extractions were performed in 96-well blocks containing 500 μl presumptive clostridia culture per well. Cells were harvested by centrifugation at 4,700 rpm for 10 minutes, the supernatant was removed. Cells were re-suspended in 500 μl of 50 mM of EDTA-2Na (pH=8.0). Aliquots of 300 μl of the suspended cells were transferred to a new 96-well block and combined with 20 μl of lysozyme from chicken egg white (Sigma, L6876) solution (100 mg/ml in 50 mM EDTA) to lyse bacterial cells. The 96-well block was incubated for 1 hour at 37° C. to lyse bacterial cells. Following the incubation 220 μl of lysis buffer (6 M Guanidine, 20% Triton-X 100, 10 mM Tris-HCL, pH 7.5) was added, mixed then incubated at room temperature for 15 minutes. Following the incubation 20 μl of Proteinase K (NEB, 800 U/mL) was added to each well, mixed and incubated at 55° C. for 30 minutes to degrade proteins. The cell lysate was then transferred to 96-well binding plate (Promega, A2278) and centrifuged at 4,700 rpms for 5 minutes. Flow through was discarded, three washes of the binding plate columns were executed centrifuging 750 μl of Column Wash Solution (Promega, A1318) at 4700 rpms for 1 minute and 30 seconds discarding flow through at the end of each spin. The binding plate was centrifuged for an additional 10 minutes at 4,700 rpm to remove any residual ethanol. A clean elution plate was then placed under the binding plate and DNA was eluted with 200 μl, pre-warmed (55° C.), Nuclease Free Water (Promega, P1195).

DNA was screened for toxin genes (α, β, ε, and ι) specific to C. perfringens using polymerase chain reaction (PCR). Amplification of toxin genes was executed using a multiplex PCR containing four primer sets (Yoo et al., 1997) (Table 1.) The PCR mixture contained 2.5 μl 10×PCR Buffer, 2 μl of 50 mM MgCl₂, 0.5 μM of each primer (Table 1.), 0.1 μl of Invitrogen™ Platinum™ Taq DNA Polymerase, 2.5 μl of DNA, sterile water was added to achieve 25 μl for a total reaction volume. The mixture underwent 5 minutes at 94° C., followed by 30 cycles of 94° C. for 1 minute, 55° C. for 1 minute, 72° C. for 1 minute finishing with a final elongation of 3 minutes at 72° C. PCR products were observed using a Fragment Analyzer (Advanced Analytics) to determine if amplification was achieved. If one or multiple toxin genes were observed a toxin type identification was assigned to each isolate based on their toxin gene profile (Songer, 1996). C. perfringens positive to total clostridia isolate ratio was used to calculate an estimated C. perfringens count based on the total clostridia count.

Unique strain-specific genetic fingerprints were generated using Random Amplification of Polymorphic DNA (RAPD) analysis on select isolates to determine diversity among fecal C. perfringens isolates. The PCR contained 5 μl of DNA, 2.5 μl RAPD primer 2 (10 μM) (Table 1.), and 17.5 μl of sterile water which was added to a Ready-To-Go RAPD Analysis Bead (Life Sciences, 27-9500-01). The mixture underwent 5 minutes at 95° C., followed by 45 cycles of 95° C. for 1 minute, 36° C. for 1 minute, 72° C. for 2 minutes finishing with a final elongation of 5 minutes at 72° C. PCR products observed on a Fragment Analyzer (Advanced Analytics) to determine amplification patterns and were imported into BioNumerics, bioinformatics software, for analysis. RAPD patterns were compared with a band based Dice correlation analysis method to determine the similarity between RAPD patterns as a way to monitor diversity between isolates.

Antimicrobial screening was done on C. perfringens isolates obtained from ruminant samples to gauge the effectiveness of the antimicrobial bacteriocin produced by the inventors' identified Bacillus strains 747, 1104, 1541, 1781, and 2018. Bacteriocin was harvested by growing each strain at 32° C. in a shaking incubator at 150 rpms for 24 hours in Brain Heart Infusion (BHI) broth. A 1% transfer of the 24-hour culture to fresh BHI broth was executed after incubation. The Bacillus were then incubated for 36-48 hours in a 32° C. shaking incubator at 150 rpms. The culture was then centrifuged at 14,000×g for 20 minutes, supernatant was then filtered with a 0.2 m filter to remove any residual cells.

A bacteriocin turbidity assay was executed by growing C. perfringens strains isolated from ruminant fecal samples in RCM for 24 hours, anaerobically, at 37° C. Overnight culture was transferred (1%) to sterile RCM and immediately used in the assay. For each C. perfringens isolate at least six wells were run in a sterile 48 well reaction plate, 600 μl inoculated culture (positive control), 600 μl inoculated RCM+70 μl bacteriocin (747, 1104, 1541, 1781, and 2018) and 670 RCM (un-inoculated, negative control). Plates were incubated anaerobically at 37° C. for 24 hours then read using a BioTek Epoch Microplate Spectrophotometer, readings were taken at a wavelength of 600 nm. Optical density readings from the negative controls were subtracted from all OD readings and percent inhibition was calculated using the positive control and each bacteriocin treatment.

To identify clostridia that did not have at least one toxin gene specific to C. perfringens, a PCR reaction was performed on the isolate DNA to amplify the 16S region of rDNA using primers 27F-YM and 1492R-Y (Table 1). This was done on 20% of the isolates that did not contain a toxin gene specific to C. perfringens. The PCR mixture contained 5 μl of 10×PCR Buffer, 2 μl of 50 mM MgCl₂, 1 μl of 50 mM dNTPs, 0.4 μM of each primer (Table 1.), 0.2 μl of Invitrogen™ Platinum™ Taq DNA Polymerase, 5 μl of DNA, and sterile water was added to achieve 50 μl for a total reaction volume. The mixture underwent 4 minutes at 95° C., followed by 35 cycles of 95° C. for 30 seconds, 50° C. for 30 seconds, 72° C. for 2 minutes finishing with a final elongation of 7 minutes at 72° C. A quality check was done on the amplification and PCR product was sent to gene wiz (https://www.genewiz.com) to obtain the sequences for the 16S genes. Sequences were compared to known typed bacterial strains obtained from EZbiocloud online electronic database (http://www.ezbiocloud.net/). Based on comparisons of these sequences a bacterial identification was assigned to the isolates.

Results:

Fecal samples, 248, were collect from 4 Upper Midwest Regional farms (Minnesota and South Dakota) from which 2,419 presumptive clostridia strains were isolated as representatives of the clostridial diversity in the Upper Midwest region (Table 13.).

Clostridia enumeration results indicated the average level of clostridia CFU/g across all calf fecal samples was 505,000 CFU/g with individual fecal samples ranging from 15 to 1,160,000 CFU/g. While the average level of clostridia CFU/g across all cow fecal samples was 8,480 CFU/g with individual fecal samples ranging from 5 to 785,000 CFU/g (FIG. 48.). Samples appeared to range in clostridia levels for both calves (FIG. 49.) and cows (FIG. 50.).

C. perfringens enumeration results displayed the average level of C. perfringens CFU/g across all calf fecal samples was 81,800 CFU/g with individual samples ranging from <10 to 1,160,000 CFU/g. While the average level of C. perfringens CFU/g across all cow fecal samples was 5,490 CFU/g with individual samples ranging from <10 to 314,000 CFU/g (FIG. 51.). Samples appeared to range in clostridia levels for both calves (FIG. 52.) and cows (FIG. 53.).

Analysis of the toxin multiplex PCR results displayed which isolates contained toxin genes specific to C. perfringens. A total of 2,419 presumptive clostridia isolates from fecal samples have been tested for the indicated C. perfringens toxin genes. Of the 2,419 clostridia isolates screened, 629 isolates (26.0%) tested positive for at least 1 of the toxin genes. From the 629 toxin-gene positive isolates 583 (92.7%) were identified as Type A (αtoxin only), however β, ε and ι, toxins were also detected in the clostridia fecal isolates.

Gentic RAPD fingerprint patterns displayed diversity among the 551 isolates that successful amplified. The isolates tested were harvested from calf fecal, cow fecal and feed and did not cluster strictly based on the sample type or farm. Isolates formed 106 clusters based on 75% similarity according to the Dice correlation method. The largest cluster was 37 isolates which was 6.7% of the total dendrogram.

Representatives from the RAPD dendrogram were selected to capture the diversity of the C. perfringens population from this region and subjected to inhibition assays. Antimicrobial testing using the bacteriocin turbidity assay displayed good inhibition of most ruminant fecal C. perfringens isolates using bacteriocin harvested from 747, 1104, 1541, 1781, and 2018. The bacteriocin from at least one of the strains 747, 1104, 1541, 1781, and 2018 were able to inhibit the growth >60% of 123 of the 135 isolates tested representing a total of 95.2% inhibition of the C. perfringens population based on the dendrogram (Table 14.).

Out of the 2,419 isolates collected 1,790 isolates (74.0%) were found to be non-toxigenic clostridia. Sequencing representatives (n=218) from the non-toxigenic clostridia displayed two dominate clostridia groups Clostridium bifermentans group (Paraclostridium bifermentans and P. benzoelyticum) and the Clostridium beijerinckii group (C. diolis, C. beijerinckii, C. chromiireducens, C. saccharoperbutylacetonicum, C. puniceum, and C. saccharobutylicum), the two main groups of the non-toxigenic clostridia group made up 50.5% of the non-toxigenic isolates (FIG. 54.).

Discussion:

Fecal samples were used as the most readily available sample type to estimate the level and obtain isolates of clostridia and C. perfringens within the digestive system of ruminants. From the 248 fecal samples collected throughout the Upper Midwest all samples had detectable levels of clostridia. Many isolates harvested (629 isolates) from the ruminant samples contained a toxin gene specific to C. perfringens. C. perfringens was detected in 74% of the cow fecal samples and in 50% of the calf fecal samples. The high presence of clostridia and C. perfringens indicates the risk for sub-acute enteric clostridia disease challenges in most ruminants throughout the Upper Midwest. C. perfringens isolates were diverse according to the RAPD genetic fingerprints but were not specific to sample type or farm. Diverse representatives of C. perfringens were mostly inhibited (>60%) by at least one bacteriocin from the following strains 747, 1104, 1541, 1781 or 2018. From the 135 isolates tested 123 isolates were inhibited by greater than 60% by at least one of the strains, representing inhibition of 95.2% of the total C. perfringens population based on representation from clusters on the RAPD dendrogram. This indicates the Bacillus strains 747, 1104, 1541, 1781 and 2018 can inhibit a wide range of diversity of C. perfringens isolates. The Bacillus strains are not limited to specific clostridia strain(s) like a vaccine which may be missing large groups of the clostridia populations based on the genetic diversity observed in the RAPD dendrogram. DNA sequencing of the non-toxigenic clostridia revealed two major identifications of Clostridium species. C. bifermentans group, which is known to produce 1,3-propanediol (Leja et al., 2014; Myszka et al., 2012) and C. beijerinckii group known to produce butanol and acetone (Hou et al., 2017). The production of the metabolic end products of these species could be having an impact in the rumen, reducing performance parameters such as milk production within a dairy cow. The high inhibition level against the clostridia isolates in vitro indicates a potential mode of action of the Bacillus strains 747, 1104, 1541, 1781 and 2018.

The Bacillus strains offer a prophylactic effect on the clostridia populations which may not only increase rumen efficiency leading to increased milk production, but prevent acute levels of C. perfringens reducing the occurrence of digestive deaths. The high prevalence of clostridia and C. perfringens in fecal samples collected suggests efficiency improvement opportunities in many ruminants throughout the Upper Midwest. This example displays the diversity of clostridia isolates from the ruminant fecal and feed samples collected from the Upper Midwest. The Bacillus strains tested 747, 1104, 1541, 1781 and 2018, could inhibit most of the clostridia diversity observed in the Upper Midwest. The product, in accordance with this embodiment of the present invention could inhibit toxigenic clostridia isolated from ruminants in the Upper Midwest indicating a benefit in rumen efficiency if fed to dairy cows as a direct fed microbial (DFM).

TABLE 13 Fecal samples, 248, were collected from Upper Midwest regional dairies separated by farm (4) and age (cow or calf) which were enumerated for clostridia, tested for C. perfringens, isolates were genotyped and tested for inhibition. Farm Name Cow Fecal Samples Calf Fecal Samples Farm BR 50 6 Farm E 45 9 Farm W 61 9 Farm Y 64 4 Total 220 28

TABLE 14 Bacteriocin assay results displaying each isolate tested and the source of that isolate, isolated from Upper Midwest regional fecal or feed samples. Inhibition was calculated based on the percent of growth for each treatment well compared to the positive control. Bacillus Strains Isolate ID Farm Type Group 747 1104 1541 1781 2018 R27.1.6 Farm E Calf Calf 99.9% 99.6% 99.6% 98.7% 99.6% R27.1.7 Farm E Calf Calf 74.3% 55.4% 58.9% 71.5% 51.2% R27.13.10 Farm E Cow Close-up 99.6% 98.5% 99.6% 99.6% 99.4% R27.13.5 Farm E Cow Close-up 99.5% 99.4% 99.4% 99.5% 99.5% R27.13.9 Farm E Cow Close-up 99.6% 99.6% 99.6% 99.8% 99.8% R27.18.8 Farm E Cow Close-up 99.9% 99.9% 99.9% 99.7% 99.9% R27.2.8 Farm E Calf Calf 99.8% 99.5% 97.0% 99.8% 99.5% R27.21.5 Farm E Cow Fresh 99.6% 99.3% 99.8% 99.6% 99.3% R27.23.3 Farm E Cow Fresh 98.5% 95.7% 97.5% 95.7% 97.5% R27.25.3 Farm E Cow Fresh 43.8% 0.0% 0.0% 6.4% 0.0% R27.26.6 Farm E Cow Fresh 99.9% 98.9% 99.9% 99.3% 99.7% R27.27.9 Farm E Cow Fresh 87.0% 0.0% 18.9% 38.5% 47.8% R27.39.9 Farm E Cow High 98.4% 95.6% 96.6% 96.6% 97.5% R27.44.5 Farm E Cow High 77.3% 0.0% 0.4% 2.4% 0.0% R27.44.6 Farm E Cow High 45.7% 25.8% 0.0% 51.6% 0.0% R27.45.1 Farm E Cow Far off 99.2% 99.5% 99.6% 98.8% 99.5% R27.45.9 Farm E Cow Far off 99.0% 98.3% 99.3% 99.0% 98.8% R27.47.1 Farm E Cow Far off 83.9% 87.5% 90.8% 88.2% 92.0% R27.48.1 Farm E Cow Far off 99.5% 99.4% 99.6% 99.5% 99.4% R27.49.4 Farm E Cow Far off 99.3% 97.4% 99.5% 99.0% 98.3% R27.50.10 Farm E Cow Far off 98.1% 98.3% 98.5% 98.0% 98.0% R27.50.6 Farm E Cow Far off 99.5% 98.0% 97.0% 99.5% 99.1% R27.50.9 Farm E Cow Far off 99.6% 99.5% 99.5% 99.7% 99.7% R27.51.7 Farm E Cow Far off 0.0% 0.0% 0.0% 0.0% 0.0% R27.53.3 Farm E Cow Far off 99.3% 90.8% 99.5% 87.6% 99.3% R27.53.5 Farm E Cow Far off 79.2% 0.0% 7.7% 16.1% 30.1% R27.54.10 Farm E Cow Far off 5.7% 0.0% 0.0% 9.4% 0.0% R27.9.1 Farm E Calf Calf 99.6% 99.4% 99.6% 99.8% 99.4% R27.9.10 Farm E Calf Calf 99.8% 99.9% 100.0% 99.9% 99.9% R27.9.2 Farm E Calf Calf 99.4% 99.8% 99.8% 99.6% 99.5% R27.9.5 Farm E Calf Calf 99.6% 99.6% 99.8% 99.6% 99.3% R31.1.7 Farm Y Calf Calf 99.8% 99.5% 99.7% 99.8% 85.6% R31.11.1 Farm Y Cow Heifer 99.6% 99.5% 99.8% 99.5% 99.5% R31.16.9 Farm Y Cow Fresh Heifer 100.0% 57.8% 38.5% 100.0% 99.9% R31.18.3 Farm Y Cow Fresh Heifer 98.4% 99.9% 99.5% 93.6% 99.9% R31.2.1 Farm Y Calf Calf 98.0% 99.2% 30.1% 99.6% 32.5% R31.22.10 Farm Y Cow Fresh Cow 62.9% 99.1% 98.8% 83.0% 99.9% R31.22.5 Farm Y Cow Fresh Cow 94.8% 97.1% 96.2% 0.0% 97.5% R31.22.8 Farm Y Cow Fresh Cow 89.6% 0.0% 99.7% 100.1% 0.0% R31.24.4 Farm Y Cow Fresh Cow 94.2% 5.4% 99.9% 68.3% 99.9% R31.25.3 Farm Y Cow Far off 0.0% 0.0% 0.0% 4.6% 0.0% R31.31.10 Farm Y Cow Far off 2.9% 0.0% 0.0% 0.0% 14.9% R31.34.8 Farm Y Cow Far off 42.1% 0.0% 0.0% 0.0% 29.0% R31.35.1 Farm Y Cow Close-up 9.6% 0.0% 0.0% 0.0% 10.3% R31.40.9 Farm Y Cow Close-up 99.8% 98.7% 99.8% 99.4% 98.3% R31.45.5 Farm Y Cow 2nd 99.9% 99.6% 99.1% 99.7% 41.4% R31.46.1 Farm Y Cow 2nd 84.4% 99.4% 99.8% 40.0% 94.1% R31.46.10 Farm Y Cow 2nd 99.6% 99.4% 100.1% 99.8% 99.6% R31.46.2 Farm Y Cow 2nd 99.5% 0.0% 99.2% 99.5% 98.6% R31.47.2 Farm Y Cow 2nd 99.7% 99.4% 99.8% 99.8% 99.5% R31.48.1 Farm Y Cow 2nd 99.5% 3.7% 99.5% 99.5% 99.4% R31.48.8 Farm Y Cow 2nd 98.0% 0.0% 99.0% 98.3% 99.2% R31.49.3 Farm Y Cow 2nd 99.2% 99.5% 99.5% 99.3% 99.2% R31.5.3 Farm Y Cow Heifer 87.0% 0.0% 0.0% 86.2% 0.0% R31.51.8 Farm Y Cow 2nd 47.6% 99.6% 99.6% 20.9% 99.3% R31.52.2 Farm Y Cow 2nd 98.7% 0.0% 99.1% 16.9% 24.0% R31.53.1 Farm Y Cow 2nd 0.0% 0.0% 0.0% 0.0% 0.0% R31.53.7 Farm Y Cow 2nd 37.1% 0.0% 83.2% 62.0% 24.6% R31.54.2 Farm Y Cow 2nd 98.9% 0.3% 28.3% 98.6% 53.4% R31.54.3 Farm Y Cow 2nd 99.4% 0.3% 27.4% 98.8% 56.9% R31.55.5 Farm Y Cow Low 67.9% 0.0% 95.3% 0.0% 32.0% R31.55.7 Farm Y Cow Low 99.1% 99.5% 99.5% 99.1% 99.5% R31.55.9 Farm Y Cow Low 36.7% 0.0% 99.5% 1.1% 5.6% R31.56.2 Farm Y Cow Low 99.2% 25.7% 99.6% 45.0% 68.5% R31.57.4 Farm Y Cow Low 99.2% 36.9% 99.5% 99.0% 99.2% R31.58.9 Farm Y Cow Low 96.6% 12.2% 92.6% 97.1% 97.4% R31.6.2 Farm Y Cow Heifer 100.0% 99.7% 99.9% 99.9% 31.4% R31.6.7 Farm Y Cow Heifer 99.0% 99.6% 99.6% 99.4% 99.6% R31.60.7 Farm Y Cow High Mature 99.0% 99.0% 99.3% 99.2% 97.5% R31.62.1 Farm Y Cow High Mature 99.1% 95.6% 99.1% 96.2% 98.1% R31.62.10 Farm Y Cow High Mature 99.7% 99.3% 99.6% 99.8% 99.4% R31.62.7 Farm Y Cow High Mature 99.5% 98.7% 99.5% 99.5% 99.5% R31.63.3 Farm Y Cow High Mature 99.2% 99.3% 99.5% 99.3% 99.3% R31.63.8 Farm Y Cow High Mature 9.6% 94.0% 99.7% 99.0% 98.0% R31.65.10 Farm Y Cow High Mature 99.5% 99.5% 99.6% 99.5% 99.4% R31.65.8 Farm Y Cow High Mature 99.5% 99.5% 99.5% 99.5% 99.5% R31.68.2 Farm Y Cow High Mature 99.4% 0.0% 78.8% 71.0% 78.0% R31.7.10 Farm Y Cow Heifer 99.7% 99.5% 99.7% 99.7% 99.7% R31.7.3 Farm Y Cow Heifer 99.4% 99.4% 99.6% 99.6% 99.3% R31.8.1 Farm Y Cow Heifer 99.5% 99.3% 99.5% 99.5% 99.3% R31.8.7 Farm Y Cow Heifer 99.3% 99.3% 99.4% 99.4% 99.4% R32.10.6 Farm W Cow Preg Mature 99.2% 99.4% 99.4% 99.6% 99.5% R32.22.7 Farm W Cow Preg Heifer 99.3% 99.3% 99.5% 99.3% 99.1% R32.32.8 Farm W Cow Close-up 99.6% 99.3% 99.6% 99.3% 99.4% R32.34.3 Farm W Cow Fresh 99.4% 98.8% 99.2% 99.4% 99.0% R32.34.9 Farm W Cow Fresh 99.0% 99.2% 99.3% 99.2% 99.2% R32.36.3 Farm W Cow Fresh 98.7% 99.1% 99.1% 97.2% 13.9% R32.39.3 Farm W Cow Fresh 99.6% 99.5% 99.6% 99.6% 99.5% R32.4.3 Farm W Calf Calf 98.9% 98.6% 99.2% 98.9% 98.6% R32.40.7 Farm W Cow Fresh 99.2% 98.9% 59.5% 99.2% 99.2% R32.44.1 Farm W Cow 1st High 99.4% 99.4% 99.4% 99.4% 99.2% R32.45.2 Farm W Cow 1st High 99.6% 13.1% 25.5% 99.5% 40.6% R32.45.5 Farm W Cow 1st High 0.0% 0.0% 0.0% 0.0% 0.0% R32.48.6 Farm W Cow 1st High 99.3% 98.3% 99.5% 99.3% 99.3% R32.48.9 Farm W Cow 1st High 99.3% 98.8% 99.1% 99.1% 99.1% R32.49.6 Farm W Cow 1st High 81.5% 54.9% 53.1% 88.7% 58.1% R32.51.2 Farm W Cow High Mature 99.4% 99.0% 99.4% 99.4% 99.5% R32.7.8 Farm W Calf Calf 99.6% 99.3% 99.4% 99.3% 99.3% R33.12.5 Farm BR Cow 1st High 99.0% 99.0% 96.5% 99.2% 99.0% R33.14.8 Farm BR Cow 1st High 99.4% 99.4% 99.7% 99.6% 99.7% R33.15.6 Farm BR Cow 1st High 99.5% 99.2% 99.8% 99.5% 99.3% R33.16.10 Farm BR Cow Fresh 99.2% 99.2% 99.2% 99.2% 99.0% R33.16.2 Farm BR Cow Fresh 99.5% 99.4% 99.5% 99.5% 99.4% R33.23.1 Farm BR Cow Fresh 99.3% 99.2% 99.3% 99.3% 99.2% R33.23.2 Farm BR Cow Fresh 99.4% 99.1% 99.3% 99.4% 99.3% R33.23.7 Farm BR Cow Fresh 99.2% 99.6% 99.6% 99.4% 99.6% R33.23.9 Farm BR Cow Fresh 99.0% 99.2% 99.4% 99.2% 99.0% R33.25.3 Farm BR Cow Fresh 99.5% 99.5% 99.6% 99.6% 99.3% R33.26.1 Farm BR Cow High Jerseys 99.6% 99.4% 99.3% 99.6% 99.6% R33.26.3 Farm BR Cow High Jerseys 99.3% 94.9% 96.5% 99.3% 41.2% R33.27.3 Farm BR Cow High Jerseys 99.3% 99.3% 99.6% 99.6% 99.3% R33.27.4 Farm BR Cow High Jerseys 99.7% 99.5% 99.9% 99.9% 99.9% R33.27.5 Farm BR Cow High Jerseys 99.9% 99.8% 99.9% 99.9% 99.6% R33.28.2 Farm BR Cow High Jerseys 99.9% 99.9% 99.8% 99.6% 99.8% R33.29.3 Farm BR Cow High Jerseys 99.6% 99.6% 99.8% 99.6% 99.6% R33.30.1 Farm BR Cow High Jerseys 99.6% 99.0% 99.0% 99.6% 99.2% R33.32.8 Farm BR Cow High Jerseys 99.4% 99.5% 98.9% 99.5% 99.5% R33.33.10 Farm BR Cow High Jerseys 99.1% 29.2% 33.4% 66.3% 41.3% R33.33.2 Farm BR Cow High Jerseys 99.4% 99.4% 99.4% 99.4% 99.2% R33.33.4 Farm BR Cow High Jerseys 99.1% 60.9% 99.4% 99.3% 99.3% R33.33.9 Farm BR Cow High Jerseys 97.4% 37.9% 50.4% 99.2% 60.2% R33.34.1 Farm BR Cow High Jerseys 99.1% 75.6% 96.1% 99.1% 98.9% R33.34.9 Farm BR Cow High Jerseys 99.9% 100.0% 100.2% 100.4% 100.3% R33.36.5 Farm BR Cow High X's 99.5% 99.2% 99.4% 99.4% 99.2% R33.37.2 Farm BR Cow High X's 99.5% 98.1% 100.2% 100.2% 99.5% R33.38.10 Farm BR Cow High X's 99.3% 98.7% 99.7% 99.5% 99.5% R33.38.6 Farm BR Cow High X's 84.5% 48.6% 42.9% 99.2% 50.6% R33.42.1 Farm BR Cow Late 99.6% 99.5% 99.8% 99.6% 99.5% R33.42.10 Farm BR Cow Late 99.5% 99.1% 99.5% 99.5% 99.1% R33.42.2 Farm BR Cow Late 99.5% 99.5% 99.8% 99.6% 99.6% R33.42.4 Farm BR Cow Late 99.6% 99.5% 99.6% 99.5% 99.5% R33.9.1 Farm BR Cow High 1st 99.5% 99.4% 93.4% 99.7% 44.1% S73.8.1 Farm E Silage Haylage Deep 24.9% 0.0% 0.0% 25.5% 0.0% S73.8.2 Farm E Silage Haylage Deep 18.3% 0.0% 0.0% 23.7% 0.0% S77.6.3 Farm Y Silage Heifer Haylage Deep 96.3% 96.3% 96.3% 97.9% 95.5%

Example 8: Selection of Bacillus Strains to Inhibit Clostridium Perfringens and Non-Toxigenic Clostridia Isolated from Ruminant Fecal Samples. (Great Lakes)

Introduction:

Clostridium is a genus of Gram-positive, spore-forming bacteria that are common residents of the gastrointestinal tract. A number of Clostridium species have been linked to enteric disease in ruminants including hemorrhagic bowel syndrome (HBS), a disease often correlated to elevated levels of Clostridium perfringens Type A. While most of the enteric diseases caused by clostridia are acute and occur sporadically in herds, in general, the prognosis is poor and the first sign of illness may be death. Based on recent results sub-acute enteric clostridia disease challenges may be a more wide spread issue than acute challenges. Due to a low success rate from treatment in acute disease challenges a more common, emphasis needs to be placed on prophylactic measures.

The purpose of this research was to characterize the distribution and diversity of clostridia in ruminants and ensure inhibition of these isolates using novel Bacillus strains as a method to control the clostridia populations.

Materials and Methods:

Fecal samples (265) from cows, heifers and calves gathered from 6 farms in the Great Lakes region were diluted 1:10 with sterile peptone, heat shocked for 30 minutes at 60° C., enumerated in sterile peptone and pour plated on Tryptose Sulphite Cycloserine (TSC) agar with D-cycloserine (400 mg/L) to select for clostridia species. Agar plates were incubated at 37° C. anaerobically for 24 hours. If present, isolated sulphite-reducing colonies were counted for a total clostridia count (CFU/g) and representative isolates were picked into Reinforced clostridia Medium (RCM) (Oxoid, CM0149) and incubated anaerobically for 24 hours at 37° C. After 24 hours of incubation the cultures were transferred (10%) to Brain Heart Infusion (BHI) broth (BD, 211059) and incubated anaerobically for 24 hours at 37° C.

DNA extractions were performed in 96-well blocks containing 500 μl presumptive clostridia culture per well. Cells were harvested by centrifugation at 4,700 rpm for 10 minutes, the supernatant was removed. Cells were re-suspended in 500 μl of 50 mM of EDTA-2Na (pH=8.0). Aliquots of 300 μl of the suspended cells were transferred to a new 96-well block and combined with 20 μl of lysozyme from chicken egg white (Sigma, L6876) solution (100 mg/ml in 50 mM EDTA) to lyse bacterial cells. The 96-well block was incubated for 1 hour at 37° C. to lyse bacterial cells. Following the incubation 220 μl of lysis buffer (6 M Guanidine, 20% Triton-X 100, 10 mM Tris-HCL, pH 7.5) was added, mixed then incubated at room temperature for 15 minutes. Following the incubation 20 μl of Proteinase K (NEB, 800 U/mL) was added to each well, mixed and incubated at 55° C. for 30 minutes to degrade proteins. The cell lysate was then transferred to 96-well binding plate (Promega, A2278) and centrifuged at 4,700 rpms for 5 minutes. Flow through was discarded, three washes of the binding plate columns were executed centrifuging 750 μl of Column Wash Solution (Promega, A1318) at 4700 rpms for 1 minute and 30 seconds discarding flow through at the end of each spin. The binding plate was centrifuged for an additional 10 minutes at 4,700 rpm to remove any residual ethanol. A clean elution plate was then placed under the binding plate and DNA was eluted with 200 μl, pre-warmed (55° C.), Nuclease Free Water (Promega, P1195).

DNA was screened for toxin genes (α, β, ε, and ι) specific to C. perfringens using polymerase chain reaction (PCR). Amplification of toxin genes was executed using a multiplex PCR containing four primer sets (Yoo et al., 1997) (Table 1.) The PCR mixture contained 2.5 μl 10×PCR Buffer, 2 μl of 50 mM MgCl₂, 0.5 μM of each primer (Table 1.), 0.1 μl of Invitrogen™ Platinum™ Taq DNA Polymerase, 2.5 μl of DNA, sterile water was added to achieve 25 μl for a total reaction volume. The mixture underwent 5 minutes at 94° C., followed by 30 cycles of 94° C. for 1 minute, 55° C. for 1 minute, 72° C. for 1 minute finishing with a final elongation of 3 minutes at 72° C. PCR products were observed using a Fragment Analyzer (Advanced Analytics) to determine if amplification was achieved. If one or multiple toxin genes were observed a toxin type identification was assigned to each isolate based on their toxin gene profile (Songer, 1996). C. perfringens positive to total clostridia isolate ratio was used to calculate an estimated C. perfringens count based on the total clostridia count.

Unique strain-specific genetic fingerprints were generated using Random Amplification of Polymorphic DNA (RAPD) analysis on select isolates to determine diversity among fecal C. perfringens isolates. The PCR contained 5 μl of DNA, 2.5 μl RAPD primer 2 (10 μM) (Table 1.), and 17.5 μl of sterile water which was added to a Ready-To-Go RAPD Analysis Bead (Life Sciences, 27-9500-01). The mixture underwent 5 minutes at 95° C., followed by 45 cycles of 95° C. for 1 minute, 36° C. for 1 minute, 72° C. for 2 minutes finishing with a final elongation of 5 minutes at 72° C. PCR products observed on a Fragment Analyzer (Advanced Analytics) to determine amplification patterns and were imported into BioNumerics, bioinformatics software, for analysis. RAPD patterns were compared with a band based Dice correlation analysis method to determine the similarity between RAPD patterns as a way to monitor diversity between isolates.

Antimicrobial screening was done on C. perfringens isolates obtained from ruminant samples to gauge the effectiveness of the antimicrobial bacteriocin produced by the inventors' identified Bacillus strains 747, 1104, 1541, 1781, and 2018. Bacteriocin was harvested by growing each strain at 32° C. in a shaking incubator at 150 rpms for 24 hours in Brain Heart Infusion (BHI) broth. A 1% transfer of the 24-hour culture to fresh BHI broth was executed after incubation. The Bacillus were then incubated for 36-48 hours in a 32° C. shaking incubator at 150 rpms. The culture was then centrifuged at 14,000×g for 20 minutes, supernatant was then filtered with a 0.2 m filter to remove any residual cells.

A bacteriocin turbidity assay was executed by growing C. perfringens strains isolated from ruminant fecal samples in RCM for 24 hours, anaerobically, at 37° C. Overnight culture was transferred (1%) to sterile RCM and immediately used in the assay. For each C. perfringens isolate at least six wells were run in a sterile 48 well reaction plate, 600 μl inoculated culture (positive control), 600 μl inoculated RCM+70 μl bacteriocin (747, 1104, 1541, 1781, and 2018) and 670 RCM (un-inoculated, negative control). Plates were incubated anaerobically at 37° C. for 24 hours then read using a BioTek Epoch Microplate Spectrophotometer, readings were taken at a wavelength of 600 nm. Optical density readings from the negative controls were subtracted from all OD readings and percent inhibition was calculated using the positive control and each bacteriocin treatment.

To identify clostridia that did not have at least one toxin gene specific to C. perfringens, a PCR reaction was performed on the isolate DNA to amplify the 16S region of rDNA using primers 27F-YM and 1492R-Y (Table 1). This was done on 20% of the isolates that did not contain a toxin gene specific to C. perfringens. The PCR mixture contained 5 μl of 10×PCR Buffer, 2 μl of 50 mM MgCl₂, 1 μl of 50 mM dNTPs, 0.4 μM of each primer (Table 1.), 0.2 μl of Invitrogen™ Platinum™ Taq DNA Polymerase, 5 μl of DNA, and sterile water was added to achieve 50 μl for a total reaction volume. The mixture underwent 4 minutes at 95° C., followed by 35 cycles of 95° C. for 30 seconds, 50° C. for 30 seconds, 72° C. for 2 minutes finishing with a final elongation of 7 minutes at 72° C. A quality check was done on the amplification and PCR product was sent to gene wiz (https://www.genewiz.com) to obtain the sequences for the 16S genes. Sequences were compared to known typed bacterial strains obtained from EZbiocloud online electronic database (http://www.ezbiocloud.net/). Based on comparisons of these sequences a bacterial identification was assigned to the isolates.

Results:

Fecal samples, 265, were collect from 6 Great Lakes Regional farms (Michigan, Indiana, Ohio, New York) from which 2,715 presumptive clostridia isolates were isolated as representatives of the clostridial diversity in the Great Lakes region (Table 15.).

Clostridia enumeration results indicated the average level of clostridia CFU/g across all calf fecal samples was 180,000 CFU/g with individual fecal samples ranging from 50 to 2,910,000 CFU/g. While the average level of clostridia CFU/g across all cow fecal samples was 69,800 CFU/g with individual fecal samples ranging from 10 to 125,000 CFU/g (FIG. 55.). Samples appeared to range in clostridia levels for both calves (FIG. 56.) and cows (FIG. 57.).

C. perfringens enumeration results displayed the average level of C. perfringens CFU/g across all calf fecal samples was 1,620 CFU/g with individual samples ranging from <10 to 1,450,000 CFU/g. While the average level of C. perfringens CFU/g across all cow fecal samples was 1,250 CFU/g with individual samples ranging from <10 to 125,000 CFU/g (FIG. 58.). Samples appeared to range in clostridia levels for both calves (FIG. 59.) and cows (FIG. 60.).

Analysis of the toxin multiplex PCR results displayed which isolates contained toxin genes specific to C. perfringens. A total of 2,715 presumptive clostridia isolates from fecal samples have been tested for the indicated C. perfringens toxin genes. Of the 2,715 clostridia isolates screened, 1,456 isolates (53.6%) tested positive for at least 1 of the toxin genes. From the 1,456 toxin-gene positive isolates 1,427 (98%) were identified as Type A (αtoxin only), however β, ε and ι, toxins were also detected in the clostridia fecal isolates.

Gentic RAPD fingerprint patterns displayed diversity among the 1,276 isolates that successful amplified. The isolates tested were harvested from calf fecal, cow fecal and feed and did not cluster strictly based on the sample type or farm. Isolates formed 162 clusters based on 75% similarity according to the Dice correlation method. The largest cluster was 102 isolates (8.0%) and comprised of isolates from all six farms.

Representatives from the RAPD dendrogram were selected to capture the diversity of the C. perfringens population from this region and subjected to inhibition assays. Antimicrobial testing using the bacteriocin turbidity assay displayed good inhibition of most ruminant fecal C. perfringens isolates using bacteriocin harvested from 747, 1104, 1541, 1781, and 2018. The bacteriocin from at least one of the strains 747, 1104, 1541, 1781, and 2018 were able to inhibit the growth >60% of 221 of the 254 isolates tested representing a total of 89.0% inhibition of the C. perfringens population based on the dendrogramb (Table 16).

Out of the 2,715 isolates collected 1,259 isolates (46%) were found to be non-toxigenic clostridia. Sequencing representatives (n=190) from the non-toxigenic clostridia displayed one dominate clostridia group, Clostridium bifermentans group (Paraclostridium bifermentans and P. benzoelyticum). Clostridium bifermentans made up 50.0% of the non-toxigenic isolates (FIG. 61.).

Discussion:

Fecal samples were used as the most readily available sample type to estimate the level and obtain isolates of clostridia and C. perfringens within the digestive system of ruminants. From the 265 fecal samples collected throughout the Great Lakes region all samples had detectable levels of clostridia. The majority of isolates harvested (53.6%) from the ruminant samples contained a toxin gene specific to C. perfringens. C. perfringens was detected in 86% of the cow fecal samples and in 63% of the calf fecal samples. The high presence of clostridia and C. perfringens indicates the risk for sub-acute enteric clostridia disease challenges in most ruminants throughout the Great Lakes region. C. perfringens isolates were diverse according to the RAPD genetic fingerprints but were not specific to sample type or farm. Diverse representatives of C. perfringens were mostly inhibited (>60%) by at least one bacteriocin from the following strains 747, 1104, 1541, 1781 or 2018. From the 254 isolates tested 221 isolates were inhibited by greater than 60% by at least one of the strains, representing inhibition of 89.0% of the total C. perfringens population based on representation from clusters on the RAPD dendrogram. This indicates the Bacillus strains 747, 1104, 1541, 1781 and 2018 could inhibit a wide range of diversity of C. perfringens isolates. The Bacillus strains are not limited to specific clostridia strain(s) like a vaccine which may be missing large groups of the clostridia populations based on the genetic diversity observed in the RAPD dendrogram. DNA sequencing of the non-toxigenic clostridia revealed one major identification of Clostridium species, Clostridium bifermentans. The high inhibition level against the clostridia isolates in vitro indicates a potential mode of action of the Bacillus strains 747, 1104, 1541, 1781 and 2018.

The Bacillus strains offer a prophylactic effect on the clostridia populations which may not only increase rumen efficiency leading to increased milk production, but prevent acute levels of C. perfringens reducing the occurrence of digestive deaths. The high prevalence of clostridia and C. perfringens in fecal samples collected suggests efficiency improvement opportunities in many ruminants throughout the Great Lakes region. This example displays the diversity of clostridia isolates from the ruminant fecal and feed samples collected from the Great Lakes region. The Bacillus strains tested 747, 1104, 1541, 1781 and 2018, were able to inhibit most of the clostridia diversity observed in the Great Lakes region. The product, in accordance with this embodiment of the present invention was able to inhibit toxigenic clostridia isolated from ruminants in the Great Lakes region indicating a benefit in rumen efficiency if fed to dairy cows as a direct fed microbial (DFM).

TABLE 15 Fecal samples, 265, were collected from Great Lakes regional dairies separated by farm (6) and age (cow or calf) which were enumerated for clostridia, tested for C. perfringens, isolates were genotyped and tested for inhibition. Farm Name State Cow Fecal Calf Fecal Farm HG Michigan 65 6 Farm H Indiana 55 9 Farm M Ohio 40 5 Farm U Ohio 10 4 Farm C New York 35 5 Farm P New York 25 6 Total 230 35

TABLE 16 Bacteriocin assay results displaying each isolate tested and the source of that isolate, isolated from Great Lakes regional fecal samples. Inhibition was calculated based on the percent of growth for each treatment well compared to the positive control. Bacillus Strains Isolate ID Farm Type Group 747 1104 1541 1781 2018 R12.1.2 Farm C Calf Fecal Calf 96.9% 95.8% 96.0% 95.0% 94.6% R12.1.6 Farm C Calf Fecal Calf 97.3% 95.5% 96.4% 95.0% 94.8% R12.11.1 Farm C Cow Fecal Medium 85.8% 79.9% 0.0% 66.7% 67.6% R12.11.4 Farm C Cow Fecal Medium 97.1% 94.7% 94.8% 92.9% 92.8% R12.11.9 Farm C Cow Fecal Medium 92.8% 84.9% 88.9% 84.4% 82.5% R12.12.1 Farm C Cow Fecal Medium 95.4% 93.7% 95.4% 93.3% 94.2% R12.12.3 Farm C Cow Fecal Medium 97.2% 95.8% 97.2% 96.6% 96.4% R12.12.4 Farm C Cow Fecal Medium 95.3% 94.0% 95.1% 94.6% 95.4% R12.13.3 Farm C Cow Fecal Medium 97.1% 96.4% 96.3% 95.2% 96.0% R12.13.6 Farm C Cow Fecal Medium 94.2% 95.3% 92.7% 91.2% 93.1% R12.14.4 Farm C Cow Fecal Medium 96.3% 95.4% 95.5% 94.9% 95.5% R12.14.7 Farm C Cow Fecal Medium 96.3% 95.4% 95.7% 94.0% 96.2% R12.14.9 Farm C Cow Fecal Medium 96.1% 87.7% 93.3% 92.6% 78.7% R12.15.7 Farm C Cow Fecal Medium 95.9% 95.3% 95.3% 94.4% 95.5% R12.16.1 Farm C Cow Fecal High 95.0% 98.6% 100.0% 100.0% 100.0% R12.16.5 Farm C Cow Fecal High 49.5% 66.3% 74.7% 70.9% 70.4% R12.17.10 Farm C Cow Fecal High 96.4% 95.9% 96.5% 95.4% 96.5% R12.18.1 Farm C Cow Fecal High 60.5% 71.8% 66.6% 66.2% 85.0% R12.18.8 Farm C Cow Fecal High 97.2% 96.4% 96.8% 96.4% 97.2% R12.18.9 Farm C Cow Fecal High 50.8% 12.1% 45.5% 55.6% 67.0% R12.19.3 Farm C Cow Fecal High 0.0% 0.0% 4.6% 2.3% 0.0% R12.19.4 Farm C Cow Fecal High 90.0% 85.6% 89.5% 83.7% 84.5% R12.20.3 Farm C Cow Fecal High 95.8% 94.1% 94.9% 94.4% 95.7% R12.20.5 Farm C Cow Fecal High 98.5% 98.5% 96.8% 95.2% 98.3% R12.21.1 Farm C Cow Fecal High 97.0% 96.2% 96.8% 96.5% 97.0% R12.21.2 Farm C Cow Fecal High 93.8% 94.8% 92.6% 91.5% 92.8% R12.21.3 Farm C Cow Fecal High 91.3% 0.0% 0.0% 18.9% 0.0% R12.21.4 Farm C Cow Fecal High 96.6% 94.8% 96.1% 93.8% 94.6% R12.21.5 Farm C Cow Fecal High 97.2% 95.2% 96.2% 94.7% 95.5% R12.22.4 Farm C Cow Fecal High 98.7% 99.0% 98.9% 98.4% 98.4% R12.22.6 Farm C Cow Fecal High 98.7% 97.9% 17.5% 98.4% 97.9% R12.24.1 Farm C Cow Fecal High 0.0% 0.0% 0.0% 0.0% 0.0% R12.24.10 Farm C Cow Fecal High 99.1% 98.7% 99.3% 98.8% 98.8% R12.24.7 Farm C Cow Fecal High 98.3% 98.1% 98.8% 97.9% 98.1% R12.24.9 Farm C Cow Fecal High 98.6% 97.7% 98.6% 98.2% 98.2% R12.25.2 Farm C Cow Fecal High 92.8% 99.7% 96.4% 92.9% 100.0% R12.25.3 Farm C Cow Fecal High 95.4% 97.8% 100.0% 96.6% 100.0% R12.25.5 Farm C Cow Fecal High 100.0% 100.0% 100.0% 100.0% 100.0% R12.25.8 Farm C Cow Fecal High 95.4% 93.1% 96.0% 96.0% 94.0% R12.25.9 Farm C Cow Fecal High 98.5% 22.5% 97.8% 98.7% 19.9% R12.26.2 Farm C Cow Fecal Far off dry 95.0% 94.2% 95.1% 95.3% 91.2% R12.26.3 Farm C Cow Fecal Far off dry 85.3% 95.6% 46.5% 85.5% 95.9% R12.26.8 Farm C Cow Fecal Far off dry 97.3% 96.7% 96.7% 96.2% 95.7% R12.26.9 Farm C Cow Fecal Far off dry 97.6% 96.8% 97.9% 98.0% 97.0% R12.27.7 Farm C Cow Fecal Far off dry 98.2% 98.5% 99.1% 98.1% 98.1% R12.28.2 Farm C Cow Fecal Far off dry 91.0% 89.1% 92.9% 89.6% 90.0% R12.29.5 Farm C Cow Fecal Far off dry 100.0% 97.4% 100.0% 100.0% 100.0% R12.29.7 Farm C Cow Fecal Far off dry 100.0% 98.6% 100.0% 100.0% 98.7% R12.3.1 Farm C Calf Fecal Calf 96.8% 93.4% 94.0% 92.3% 92.0% R12.3.6 Farm C Calf Fecal Calf 97.1% 95.0% 94.4% 93.6% 92.5% R12.3.7 Farm C Calf Fecal Calf 0.0% 0.0% 0.0% 0.0% 0.0% R12.3.9 Farm C Calf Fecal Calf 8.1% 0.0% 0.0% 0.0% 0.0% R12.30.5 Farm C Cow Fecal Far off dry 98.7% 98.1% 98.7% 98.1% 98.1% R12.30.6 Farm C Cow Fecal Far off dry 98.5% 97.4% 98.3% 98.7% 98.1% R12.32.4 Farm C Cow Fecal Fresh 95.3% 89.3% 94.7% 94.0% 100.7% R12.32.8 Farm C Cow Fecal Fresh 99.1% 98.8% 99.7% 98.3% 98.8% R12.33.10 Farm C Cow Fecal Fresh 98.9% 98.5% 99.4% 98.5% 98.7% R12.34.3 Farm C Cow Fecal Fresh 99.2% 98.3% 99.1% 98.5% 98.4% R12.37.3 Farm C Cow Fecal Close Up 98.7% 98.6% 99.1% 98.5% 98.2% R12.38.10 Farm C Cow Fecal Close Up 96.5% 95.1% 96.9% 95.3% 95.1% R12.4.2 Farm C Calf Fecal Calf 57.7% 39.7% 0.0% 17.9% 2.6% R12.4.4 Farm C Calf Fecal Calf 0.0% 0.0% 0.0% 0.0% 0.0% R12.4.9 Farm C Calf Fecal Calf 95.4% 93.5% 92.5% 91.0% 90.1% R12.40.2 Farm C Cow Fecal Close Up 74.1% 71.6% 80.2% 76.5% 72.8% R12.5.1 Farm C Calf Fecal Calf 97.0% 94.9% 18.1% 93.7% 59.8% R12.5.2 Farm C Calf Fecal Calf 77.8% 51.5% 58.6% 34.3% 32.3% R12.5.5 Farm C Calf Fecal Calf 84.9% 75.3% 71.0% 65.6% 64.5% R12.5.8 Farm C Calf Fecal Calf 55.3% 0.0% 6.4% 0.0% 0.0% R12.6.10 Farm C Cow Fecal Medium 96.7% 95.8% 95.8% 94.9% 95.5% R12.7.2 Farm C Cow Fecal Medium 97.7% 96.8% 96.6% 95.9% 96.5% R12.7.7 Farm C Cow Fecal Medium 95.4% 92.2% 91.9% 89.8% 87.6% R12.9.2 Farm C Cow Fecal Medium 97.3% 96.4% 95.9% 95.4% 95.9% R12.9.3 Farm C Cow Fecal Medium 95.6% 92.7% 92.4% 89.5% 89.2% R13.1.5 Farm P Calf Fecal Calf 95.5% 92.7% 31.7% 94.7% 28.6% R13.1.8 Farm P Calf Fecal Calf 93.2% 59.4% 0.0% 93.7% 1.0% R13.10.10 Farm P Cow Fecal Robot North 96.3% 95.5% 81.0% 95.1% 91.4% R13.10.3 Farm P Cow Fecal Robot North 96.1% 94.3% 91.3% 88.4% 18.6% R13.10.6 Farm P Cow Fecal Robot North 98.9% 98.6% 99.2% 98.4% 98.5% R13.10.9 Farm P Cow Fecal Robot North 99.1% 61.1% 39.8% 97.4% 63.1% R13.11.2 Farm P Cow Fecal Robot North 94.7% 88.6% 90.9% 92.1% 89.7% R13.11.6 Farm P Cow Fecal Robot North 9.7% 15.9% 20.9% 6.1% 29.7% R13.11.8 Farm P Cow Fecal Robot North 27.3% 93.9% 0.0% 95.0% 4.3% R13.11.9 Farm P Cow Fecal Robot North 96.2% 95.6% 95.7% 95.2% 96.0% R13.12.10 Farm P Cow Fecal Robot South 59.1% 5.1% 0.0% 80.0% 43.7% R13.12.3 Farm P Cow Fecal Robot South 95.1% 94.1% 70.9% 93.7% 92.9% R13.13.4 Farm P Cow Fecal Robot South 96.2% 0.6% 0.0% 97.7% 29.9% R13.13.8 Farm P Cow Fecal Robot South 77.5% 42.9% 0.6% 69.9% 15.8% R13.14.3 Farm P Cow Fecal Robot South 60.0% 51.7% 12.3% 68.7% 35.8% R13.15.4 Farm P Cow Fecal Robot South 89.3% 69.4% 35.5% 89.2% 76.0% R13.16.5 Farm P Cow Fecal Far Dry 96.3% 28.3% 7.5% 95.9% 11.7% R13.16.7 Farm P Cow Fecal Far Dry 49.2% 16.1% 1.8% 54.5% 4.3% R13.16.9 Farm P Cow Fecal Far Dry 95.0% 14.5% 5.1% 97.8% 35.0% R13.17.1 Farm P Cow Fecal Far Dry 88.1% 15.2% 0.0% 85.7% 64.8% R13.17.5 Farm P Cow Fecal Far Dry 0.0% 0.0% 0.0% 0.0% 0.0% R13.18.3 Farm P Cow Fecal Far Dry 0.0% 0.0% 0.0% 0.0% 0.0% R13.19.4 Farm P Cow Fecal Far Dry 73.2% 72.5% 8.4% 73.7% 61.1% R13.2.2 Farm P Calf Fecal Calf 92.0% 3.4% 0.0% 91.3% 1.5% R13.2.3 Farm P Calf Fecal Calf 99.2% 99.0% 99.4% 99.6% 98.3% R13.21.1 Farm P Cow Fecal Far Dry 95.4% 14.0% 0.0% 95.0% 0.0% R13.21.5 Farm P Cow Fecal Far Dry 5.7% 12.7% 16.0% 0.0% 12.0% R13.22.7 Farm P Cow Fecal 1st Lact 0.0% 5.2% 5.6% 0.0% 10.8% R13.23.2 Farm P Cow Fecal 1st Lact 0.0% 0.0% 0.8% 0.0% 15.9% R13.23.5 Farm P Cow Fecal 1st Lact 55.5% 45.5% 17.6% 66.3% 46.5% R13.23.7 Farm P Cow Fecal 1st Lact 96.8% 31.5% 48.1% 47.5% 39.5% R13.25.5 Farm P Cow Fecal 1st Lact 78.5% 45.9% 10.3% 76.3% 60.3% R13.25.6 Farm P Cow Fecal 1st Lact 86.0% 23.1% 0.0% 6.5% 48.3% R13.25.9 Farm P Cow Fecal 1st Lact 93.5% 85.0% 38.8% 94.5% 29.8% R13.26.4 Farm P Cow Fecal 1st Lact 83.4% 57.0% 20.2% 89.0% 61.4% R13.26.5 Farm P Cow Fecal 1st Lact 72.9% 62.8% 0.0% 80.5% 51.9% R13.27.7 Farm P Cow Fecal Close-up Dry 42.0% 0.0% 7.2% 28.8% 36.8% R13.28.1 Farm P Cow Fecal Close-up Dry 75.7% 63.3% 0.0% 85.8% 52.9% R13.28.2 Farm P Cow Fecal Close-up Dry 88.8% 32.6% 16.2% 27.5% 57.7% R13.31.4 Farm P Cow Fecal Close-up Dry 92.9% 28.6% 25.5% 99.7% 51.0% R13.4.1 Farm P Calf Fecal Calf 93.8% 61.8% 90.8% 88.9% 17.3% R13.5.10 Farm P Calf Fecal Calf 94.1% 91.6% 7.2% 93.9% 17.8% R13.5.3 Farm P Calf Fecal Calf 96.7% 95.9% 96.1% 96.0% 96.5% R13.5.5 Farm P Calf Fecal Calf 89.0% 85.3% 54.0% 83.4% 55.7% R13.5.7 Farm P Calf Fecal Calf 96.4% 95.3% 94.0% 93.0% 93.7% R13.5.8 Farm P Calf Fecal Calf 95.1% 50.9% 51.4% 91.7% 20.5% R13.6.10 Farm P Calf Fecal Calf 93.7% 93.5% 91.2% 76.5% 85.4% R13.7.5 Farm P Cow Fecal Robot North 22.5% 3.0% 0.0% 85.4% 6.4% R13.7.7 Farm P Cow Fecal Robot North 95.9% 95.2% 96.3% 95.4% 96.1% R13.8.3 Farm P Cow Fecal Robot North 100.0% 99.7% 99.9% 100.0% 100.0% R13.9.5 Farm P Cow Fecal Robot North 95.6% 94.7% 93.7% 95.5% 95.0% R13.9.6 Farm P Cow Fecal Robot North 96.7% 95.1% 0.0% 96.3% 0.0% R13.9.8 Farm P Cow Fecal Robot North 15.9% 5.4% 10.8% 14.9% 13.9% R28.16.5 Farm HG Cow Fecal Fresh 99.7% 99.8% 99.8% 88.1% 100.0% R28.21.3 Farm HG Cow Fecal Close-up 97.4% 95.8% 99.5% 94.9% 97.2% R28.26.2 Farm HG Cow Fecal Close-up 27.0% 0.0% 0.0% 6.7% 40.4% R28.26.3 Farm HG Cow Fecal Close-up 21.0% 19.0% 0.0% 0.0% 7.6% R28.26.4 Farm HG Cow Fecal Close-up 96.3% 97.3% 90.7% 96.0% 95.6% R28.29.5 Farm HG Cow Fecal First 96.5% 95.5% 92.2% 94.8% 96.5% R28.39.3 Farm HG Cow Fecal High 98.0% 98.2% 99.9% 97.6% 98.4% R28.39.8 Farm HG Cow Fecal High 18.2% 0.0% 0.0% 0.0% 2.8% R28.42.5 Farm HG Cow Fecal High 92.2% 91.9% 88.2% 92.6% 95.7% R28.47.1 Farm HG Cow Fecal Late 100.2% 99.8% 99.8% 99.8% 100.2% R28.51.1 Farm HG Cow Fecal Far Off 99.1% 67.0% 100.0% 100.0% 78.9% R28.52.4 Farm HG Cow Fecal Far Off 74.5% 22.0% 76.7% 70.0% 71.4% R28.58.4 Farm HG Cow Fecal Far Off 100.0% 98.2% 100.0% 100.0% 99.8% R28.62.10 Farm HG Cow Fecal Negative 98.5% 40.8% 98.8% 99.2% 99.9% R28.65.4 Farm HG Cow Fecal Negative 86.1% 82.6% 80.6% 88.6% 92.1% R28.65.5 Farm HG Cow Fecal Negative 98.5% 98.8% 98.7% 98.2% 99.1% R28.65.8 Farm HG Cow Fecal Negative 100.0% 100.0% 100.0% 100.0% 99.3% R28.65.9 Farm HG Cow Fecal Negative 100.5% 99.9% 98.8% 95.9% 100.5% R28.67.5 Farm HG Cow Fecal Negative 58.8% 44.8% 67.0% 60.5% 68.6% R28.68.6 Farm HG Cow Fecal Negative 46.1% 100.0% 100.0% 94.2% 95.5% R28.7.5 Farm HG Cow Fecal Fresh 52.5% 52.5% 0.0% 39.7% 44.0% R28.7.8 Farm HG Cow Fecal Fresh 71.7% 76.3% 0.0% 79.9% 75.9% R28.70.1 Farm HG Cow Fecal Negative 100.0% 99.4% 99.9% 100.0% 100.0% R28.70.2 Farm HG Cow Fecal Negative 100.0% 99.7% 100.0% 100.0% 100.0% R28.70.3 Farm HG Cow Fecal Negative 98.0% 98.4% 97.8% 97.0% 98.4% R28.71.8 Farm HG Cow Fecal Negative 51.2% 0.0% 34.4% 36.3% 66.4% R28.8.1 Farm HG Cow Fecal Fresh 78.9% 83.5% 83.5% 78.6% 79.9% R29.1.1 Farm H Calf Fecal Calf 77.2% 78.5% 70.6% 74.7% 81.2% R29.10.6 Farm H Cow Fecal Fresh 100.0% 91.9% 99.5% 99.8% 99.8% R29.12.2 Farm H Cow Fecal Fresh 100.0% 42.8% 100.0% 98.7% 99.8% R29.16.10 Farm H Cow Fecal Fresh 99.6% 99.8% 99.8% 99.8% 99.9% R29.17.1 Farm H Cow Fecal Fresh 97.4% 85.2% 84.6% 97.7% 96.7% R29.19.5 Farm H Cow Fecal Fresh 34.7% 28.3% 26.0% 32.3% 33.4% R29.20.2 Farm H Cow Fecal Fresh 100.0% 99.7% 97.0% 98.9% 100.0% R29.21.10 Farm H Cow Fecal Fresh 100.0% 100.0% 99.4% 98.6% 100.0% R29.21.9 Farm H Cow Fecal Fresh 100.0% 100.0% 99.4% 98.8% 91.7% R29.22.1 Farm H Cow Fecal Fresh 100.0% 100.0% 99.8% 98.6% 100.0% R29.22.5 Farm H Cow Fecal Fresh 100.0% 100.0% 100.0% 99.1% 100.0% R29.22.7 Farm H Cow Fecal Fresh 100.0% 100.0% 100.0% 99.1% 99.8% R29.23.10 Farm H Cow Fecal Fresh 100.0% 100.0% 98.5% 98.5% 99.4% R29.23.8 Farm H Cow Fecal Fresh 100.0% 100.0% 98.5% 98.6% 99.6% R29.24.1 Farm H Cow Fecal Fresh 90.3% 88.0% 85.8% 89.4% 90.8% R29.24.3 Farm H Cow Fecal Fresh 100.0% 97.8% 96.3% 97.4% 98.7% R29.24.9 Farm H Cow Fecal Fresh 100.0% 99.8% 99.1% 97.2% 98.4% R29.25.7 Farm H Cow Fecal Close-up 100.0% 14.0% 99.6% 97.6% 98.7% R29.26.4 Farm H Cow Fecal Close-up 100.0% 96.9% 99.9% 96.0% 98.4% R29.26.7 Farm H Cow Fecal Close-up 100.0% 39.3% 100.0% 84.6% 96.8% R29.28.1 Farm H Cow Fecal Close-up 100.0% 100.0% 100.0% 100.0% 100.0% R29.28.7 Farm H Cow Fecal Close-up 99.6% 56.5% 37.5% 99.3% 97.7% R29.31.1 Farm H Cow Fecal Close-up 100.0% 0.0% 100.0% 0.0% 100.0% R29.31.3 Farm H Cow Fecal Close-up 99.7% 0.0% 100.0% 6.5% 100.0% R29.35.7 Farm H Cow Fecal Pregnant 100.0% 67.0% 70.9% 100.0% 99.9% R29.35.8 Farm H Cow Fecal Pregnant 100.0% 99.1% 100.0% 100.0% 99.9% R29.36.2 Farm H Cow Fecal Pregnant 100.0% 0.0% 60.5% 0.0% 0.0% R29.36.8 Farm H Cow Fecal Pregnant 99.7% 0.0% 13.3% 99.3% 98.7% R29.40.9 Farm H Cow Fecal Pregnant 74.8% 71.0% 73.1% 71.2% 70.6% R29.49.2 Farm H Cow Fecal High 100.0% 99.0% 100.0% 100.0% 100.0% R29.49.9 Farm H Cow Fecal High 61.9% 58.5% 64.7% 61.0% 62.0% R29.60.3 Farm H Cow Fecal Late 100.0% 100.0% 100.0% 100.0% 99.6% R29.63.3 Farm H Cow Fecal Late 100.0% 100.0% 100.0% 100.0% 100.0% R29.63.5 Farm H Cow Fecal Late 100.0% 99.3% 43.9% 100.0% 93.6% R29.63.6 Farm H Cow Fecal Late 100.0% 99.4% 100.0% 100.0% 100.0% R29.63.7 Farm H Cow Fecal Late 100.0% 99.1% 100.0% 100.0% 100.0% R29.63.8 Farm H Cow Fecal Late 54.9% 46.2% 45.7% 53.4% 67.5% R34.11.8 Farm U Cow Fecal Lact. Cows 100.0% 65.0% 48.3% 100.0% 82.8% R34.13.10 Farm U Cow Fecal Lact. Cows 86.1% 47.4% 38.0% 80.9% 87.9% R34.14.3 Farm U Cow Fecal Lact. Cows 97.0% 17.2% 21.9% 24.3% 61.8% R34.4.4 Farm U Calf Fecal Calf 99.9% 97.9% 99.7% 100.1% 99.2% R34.6.1 Farm U Cow Fecal Lact. Cows 84.1% 54.0% 4.3% 80.0% 81.8% R34.9.2 Farm U Cow Fecal Lact. Cows 90.2% 0.0% 0.0% 100.0% 100.0% R34.9.4 Farm U Cow Fecal Lact. Cows 71.2% 46.8% 49.5% 83.2% 31.7% R34.9.8 Farm U Cow Fecal Lact. Cows 100.0% 35.7% 100.0% 100.0% 100.0% R35.10.1 Farm M Cow Fecal Hi 2 yr olds 80.0% 1.5% 2.8% 91.9% 37.5% R35.10.5 Farm M Cow Fecal Hi 2 yr olds 34.5% 99.0% 99.6% 100.0% 99.7% R35.10.6 Farm M Cow Fecal Hi 2 yr olds 100.0% 100.0% 0.0% 100.0% 98.0% R35.10.7 Farm M Cow Fecal Hi 2 yr olds 99.8% 55.8% 59.3% 92.4% 99.1% R35.11.4 Farm M Cow Fecal Hi 2 yr olds 99.1% 97.9% 0.0% 95.9% 15.3% R35.11.6 Farm M Cow Fecal Hi 2 yr olds 99.8% 98.1% 23.3% 100.0% 0.0% R35.11.7 Farm M Cow Fecal Hi 2 yr olds 98.2% 0.0% 0.0% 37.1% 87.4% R35.11.9 Farm M Cow Fecal Hi 2 yr olds 98.6% 0.0% 90.9% 87.4% 62.0% R35.12.9 Farm M Cow Fecal Hi 2 yr olds 67.2% 28.0% 0.0% 63.0% 36.2% R35.21.9 Farm M Cow Fecal High Mature cows 92.9% 59.8% 0.0% 90.7% 10.5% R35.22.1 Farm M Cow Fecal High Mature cows 0.0% 0.0% 0.0% 0.0% 0.0% R35.25.6 Farm M Cow Fecal High Mature cows 99.8% 99.8% 99.8% 99.8% 97.1% R35.26.3 Farm M Cow Fecal Low Cows 100.0% 98.9% 100.0% 100.0% 100.0% R35.26.5 Farm M Cow Fecal Low Cows 68.4% 40.6% 58.9% 62.5% 67.7% R35.28.3 Farm M Cow Fecal Low Cows 99.8% 98.8% 22.4% 97.6% 97.2% R35.29.10 Farm M Cow Fecal Low Cows 100.0% 99.0% 100.0% 100.0% 100.0% R35.29.8 Farm M Cow Fecal Low Cows 100.0% 100.0% 98.8% 100.0% 100.0% R35.3.2 Farm M Calf Fecal Calf 100.0% 99.9% 100.0% 100.0% 100.0% R35.30.8 Farm M Cow Fecal Low Cows 100.0% 99.1% 100.0% 100.0% 100.0% R35.31.2 Farm M Cow Fecal Low Cows 99.8% 98.3% 98.3% 99.7% 99.5% R35.32.10 Farm M Cow Fecal Low Cows 87.2% 84.2% 82.4% 96.0% 88.9% R35.33.5 Farm M Cow Fecal Low Cows 22.8% 0.0% 18.4% 0.0% 0.0% R35.35.2 Farm M Cow Fecal Dry Cows 100.0% 84.1% 100.0% 100.0% 100.0% R35.35.5 Farm M Cow Fecal Dry Cows 96.4% 88.3% 91.5% 91.6% 93.1% R35.35.8 Farm M Cow Fecal Dry Cows 100.0% 99.6% 100.0% 97.7% 100.0% R35.38.9 Farm M Cow Fecal Dry Cows 96.3% 98.3% 98.1% 95.5% 98.7% R35.6.6 Farm M Cow Fecal Hi 2 yr olds 100.0% 99.6% 99.9% 100.0% 100.0% R35.6.9 Farm M Cow Fecal Hi 2 yr olds 91.2% 0.0% 0.0% 96.0% 43.9% R35.8.2 Farm M Cow Fecal Hi 2 yr olds 100.0% 100.0% 100.0% 100.0% 100.0% R35.8.3 Farm M Cow Fecal Hi 2 yr olds 100.0% 6.1% 0.0% 100.0% 100.0% R35.9.5 Farm M Cow Fecal Hi 2 yr olds 100.0% 100.0% 11.7% 100.0% 100.0% S52.14.1 Farm P TMR Robot Bam 0.0% 0.0% 11.8% 20.6% 69.0% S52.14.7 Farm P TMR Robot Bam 0.0% 0.0% 0.0% 0.0% 0.0% S53.11.1 Farm C TMR Fresh 0.0% 0.0% 0.0% 0.0% 0.0% S53.5.5 Farm C Straw Dry Cows 0.0% 0.0% 0.0% 0.0% 2.1% S53.5.6 Farm C Straw Dry Cows 32.6% 35.9% 9.1% 41.1% 40.7% S53.5.7 Farm C Straw Dry Cows 0.0% 0.0% 0.0% 0.0% 0.0% S53.7.3 Farm C Ground Corn 0.0% 6.4% 0.0% 7.3% 0.0% S53.7.6 Farm C Ground Corn 0.0% 0.0% 0.0% 0.0% 0.0% S53.7.7 Farm C Ground Corn 10.9% 0.0% 0.0% 0.0% 8.4% S74.7.1 Farm HG Ground Corn 73.9% 65.9% 46.1% 35.3% 65.9% S75.3.1 Farm H TMR Close-up 100.0% 99.2% 100.0% 100.0% 100.0% S75.3.10 Farm H TMR Close-up 100.0% 100.0% 100.0% 100.0% 100.0% S75.5.1 Farm H TMR Late 34.4% 24.2% 40.7% 60.0% 43.1% S75.8.1 Farm H Pellets Calf 100.0% 100.0% 100.0% 100.0% 100.0% S75.8.3 Farm H Pellets Calf 100.0% 99.8% 99.7% 100.0% 100.0% S75.8.5 Farm H Pellets Calf 100.0% 100.0% 100.0% 100.0% 99.2% S75.8.9 Farm H Pellets Calf 100.0% 100.0% 100.0% 100.0% 100.0% S75.9.1 Farm H Soy Hulls 100.0% 100.0% 99.7% 100.0% 99.7% S75.9.2 Farm H Soy Hulls 99.7% 100.0% 99.5% 99.8% 99.8% S80.4.1 Farm U TMR Cow 0.0% 0.0% 0.0% 33.7% 13.4% S81.10.6 Farm M Wheat Midds 100.0% 100.0% 100.0% 100.0% 100.0% S81.11.1 Farm M Bird Seed Screening 91.5% 82.3% 91.1% 83.0% 80.1% S81.11.7 Farm M Bird Seed Screening 30.5% 32.9% 22.6% 37.2% 27.1% S81.3.6 Farm M Haylage 3rd 25.4% 0.0% 0.0% 0.0% 0.0% S81.7.4 Farm M TMR Cow 94.5% 90.9% 89.5% 92.4% 92.0%

Example 9: Selection of Bacillus Strains to Inhibit Clostridium Perfringens and Non-Toxigenic Clostridia Isolated from Ruminant Fecal Samples. (North East)

Introduction:

Clostridium is a genus of Gram-positive, spore-forming bacteria that are common residents of the gastrointestinal tract. A number of Clostridium species have been linked to enteric disease in ruminants including hemorrhagic bowel syndrome (HBS), a disease often correlated to elevated levels of Clostridium perfringens Type A. While most of the enteric diseases caused by clostridia are acute and occur sporadically in herds, in general, the prognosis is poor and the first sign of illness may be death. Based on recent results sub-acute enteric clostridia disease challenges may be a more wide spread issue than acute challenges. Due to a low success rate from treatment in acute disease challenges a more common, emphasis needs to be placed on prophylactic measures.

The purpose of this research was to characterize the distribution and diversity of clostridia in ruminants and ensure inhibition of these isolates using novel Bacillus strains as a method to control the clostridia populations.

Materials and Methods:

Fecal samples (339) from cows, heifers and calves gathered from 9 farms in the Northeast region were diluted 1:10 with sterile peptone, heat shocked for 30 minutes at 60° C., enumerated in sterile peptone and pour plated on Tryptose Sulphite Cycloserine (TSC) agar with D-cycloserine (400 mg/L) to select for clostridia species. Agar plates were incubated at 37° C. anaerobically for 24 hours. If present, isolated sulphite-reducing colonies were counted for a total clostridia count (CFU/g) and representative isolates were picked into Reinforced clostridia Medium (RCM) (Oxoid, CM0149) and incubated anaerobically for 24 hours at 37° C. After 24 hours of incubation the cultures were transferred (10%) to Brain Heart Infusion (BHI) broth (BD, 211059) and incubated anaerobically for 24 hours at 37° C.

DNA extractions were performed in 96-well blocks containing 500 μl presumptive clostridia culture per well. Cells were harvested by centrifugation at 4,700 rpm for 10 minutes, the supernatant was removed. Cells were re-suspended in 500 μl of 50 mM of EDTA-2Na (pH=8.0). Aliquots of 300 μl of the suspended cells were transferred to a new 96-well block and combined with 20 μl of lysozyme from chicken egg white (Sigma, L6876) solution (100 mg/ml in 50 mM EDTA) to lyse bacterial cells. The 96-well block was incubated for 1 hour at 37° C. to lyse bacterial cells. Following the incubation 220 μl of lysis buffer (6 M Guanidine, 20% Triton-X 100, 10 mM Tris-HCL, pH 7.5) was added, mixed then incubated at room temperature for 15 minutes. Following the incubation 20 μl of Proteinase K (NEB, 800 U/mL) was added to each well, mixed and incubated at 55° C. for 30 minutes to degrade proteins. The cell lysate was then transferred to 96-well binding plate (Promega, A2278) and centrifuged at 4,700 rpms for 5 minutes. Flow through was discarded, three washes of the binding plate columns were executed centrifuging 750 μl of Column Wash Solution (Promega, A1318) at 4700 rpms for 1 minute and 30 seconds discarding flow through at the end of each spin. The binding plate was centrifuged for an additional 10 minutes at 4,700 rpm to remove any residual ethanol. A clean elution plate was then placed under the binding plate and DNA was eluted with 200 μl, pre-warmed (55° C.), Nuclease Free Water (Promega, P1195).

DNA was screened for toxin genes (α, β, ε, and ι) specific to C. perfringens using polymerase chain reaction (PCR). Amplification of toxin genes was executed using a multiplex PCR containing four primer sets (Yoo et al., 1997) (Table 1.) The PCR mixture contained 2.5 μl 10×PCR Buffer, 2 μl of 50 mM MgCl₂, 0.5 μM of each primer (Table 1.), 0.1 μl of Invitrogen™ Platinum™ Taq DNA Polymerase, 2.5 μl of DNA, sterile water was added to achieve 25 μl for a total reaction volume. The mixture underwent 5 minutes at 94° C., followed by 30 cycles of 94° C. for 1 minute, 55° C. for 1 minute, 72° C. for 1 minute finishing with a final elongation of 3 minutes at 72° C. PCR products were observed using a Fragment Analyzer (Advanced Analytics) to determine if amplification was achieved. If one or multiple toxin genes were observed a toxin type identification was assigned to each isolate based on their toxin gene profile (Songer, 1996). C. perfringens positive to total clostridia isolate ratio was used to calculate an estimated C. perfringens count based on the total clostridia count.

Unique strain-specific genetic fingerprints were generated using Random Amplification of Polymorphic DNA (RAPD) analysis on select isolates to determine diversity among fecal C. perfringens isolates. The PCR contained 5 μl of DNA, 2.5 μl RAPD primer 2 (10 μM) (Table 1.), and 17.5 μl of sterile water which was added to a Ready-To-Go RAPD Analysis Bead (Life Sciences, 27-9500-01). The mixture underwent 5 minutes at 95° C., followed by 45 cycles of 95° C. for 1 minute, 36° C. for 1 minute, 72° C. for 2 minutes finishing with a final elongation of 5 minutes at 72° C. PCR products observed on a Fragment Analyzer (Advanced Analytics) to determine amplification patterns and were imported into BioNumerics, bioinformatics software, for analysis. RAPD patterns were compared with a band based Dice correlation analysis method to determine the similarity between RAPD patterns as a way to monitor diversity between isolates.

Antimicrobial screening was done on C. perfringens isolates obtained from ruminant samples to gauge the effectiveness of the antimicrobial bacteriocin produced by the inventors' identified Bacillus strains 747, 1104, 1541, 1781, and 2018. Bacteriocin was harvested by growing each strain at 32° C. in a shaking incubator at 150 rpms for 24 hours in Brain Heart Infusion (BHI) broth. A 1% transfer of the 24-hour culture to fresh BHI broth was executed after incubation. The Bacillus were then incubated for 36-48 hours in a 32° C. shaking incubator at 150 rpms. The culture was then centrifuged at 14,000×g for 20 minutes, supernatant was then filtered with a 0.2 m filter to remove any residual cells.

A bacteriocin turbidity assay was executed by growing C. perfringens strains isolated from ruminant fecal samples in RCM for 24 hours, anaerobically, at 37° C. Overnight culture was transferred (1%) to sterile RCM and immediately used in the assay. For each C. perfringens isolate at least six wells were run in a sterile 48 well reaction plate, 600 μl inoculated culture (positive control), 600 μl inoculated RCM+70 μl bacteriocin (747, 1104, 1541, 1781, and 2018) and 670 RCM (un-inoculated, negative control). Plates were incubated anaerobically at 37° C. for 24 hours then read using a BioTek Epoch Microplate Spectrophotometer, readings were taken at a wavelength of 600 nm. Optical density readings from the negative controls were subtracted from all OD readings and percent inhibition was calculated using the positive control and each bacteriocin treatment.

To identify clostridia that did not have at least one toxin gene specific to C. perfringens, a PCR reaction was performed on the isolate DNA to amplify the 16S region of rDNA using primers 27F-YM and 1492R-Y (Table 1). This was done on 20% of the isolates that did not contain a toxin gene specific to C. perfringens. The PCR mixture contained 5 μl of 10×PCR Buffer, 2 μl of 50 mM MgCl₂, 1 μl of 50 mM dNTPs, 0.4 μM of each primer (Table 1.), 0.2 μl of Invitrogen™ Platinum™ Taq DNA Polymerase, 5 μl of DNA, and sterile water was added to achieve 50 μl for a total reaction volume. The mixture underwent 4 minutes at 95° C., followed by 35 cycles of 95° C. for 30 seconds, 50° C. for 30 seconds, 72° C. for 2 minutes finishing with a final elongation of 7 minutes at 72° C. A quality check was done on the amplification and PCR product was sent to gene wiz (https://www.genewiz.com) to obtain the sequences for the 16S genes. Sequences were compared to known typed bacterial strains obtained from EZbiocloud online electronic database (http://www.ezbiocloud.net/). Based on comparisons of these sequences a bacterial identification was assigned to the isolates.

Results:

Fecal samples, 339, were collect from 9 Northeast regional farms (Maine, Vermont, and New York) from which 3,252 presumptive clostridia isolates were isolated as representatives of the clostridial diversity in the Northeast region (Table 17.).

Clostridia enumeration results indicated the average level of clostridia CFU/g across all calf fecal samples was 466,000 CFU/g with individual fecal samples ranging from 25 to 1,250,000 CFU/g. While the average level of clostridia CFU/g across all cow fecal samples was 18,200 CFU/g with individual fecal samples ranging from 5 to 3,540,000 CFU/g (FIG. 62.). Samples appeared to range in clostridia levels for both calves (FIG. 63.) and cows (FIG. 64.).

C. perfringens enumeration results displayed the average level of C. perfringens CFU/g across all calf fecal samples was 311,000 CFU/g with individual samples ranging from <10 to 3,540,000 CFU/g. While the average level of C. perfringens CFU/g across all cow fecal samples was 16,700 CFU/g with individual samples ranging from <10 to 3,540,000 CFU/g (FIG. 65.). Samples appeared to range in clostridia levels for both calves (FIG. 66.) and cows (FIG. 67.).

Analysis of the toxin multiplex PCR results displayed which isolates contained toxin genes specific to C. perfringens. A total of 3,252 presumptive clostridia isolates from fecal samples have been tested for the indicated C. perfringens toxin genes. Of the 3,252 clostridia isolates screened, 1,795 isolates (55.2%) tested positive for at least 1 of the toxin genes. From the 1,795 toxin-gene positive isolates 1,687 (94%) were identified as Type A (αtoxin only), however β, ε and ι, toxins were also detected in the clostridia fecal isolates.

Gentic RAPD fingerprint patterns displayed diversity among the 1,619 isolates that successful amplified. The isolates tested were harvested from calf fecal, cow fecal and feed and did not cluster strictly based on the sample type or farm. Isolates formed 361 clusters based on 75% similarity according to the Dice correlation method. The largest cluster was 82 isolates (5.1%) and comprised of isolates from several farms.

Representatives from the RAPD dendrogram were selected to capture the diversity of the C. perfringens population from this region and subjected to inhibition assays. Antimicrobial testing using the bacteriocin turbidity assay displayed good inhibition of most ruminant fecal C. perfringens isolates using bacteriocin harvested from 747, 1104, 1541, 1781, and 2018. The bacteriocin from at least one of the strains 747, 1104, 1541, 1781, and 2018 were able to inhibit the growth >60% of 342 of the 412 isolates tested representing a total of 84.6% inhibition of the C. perfringens population based on the dendrogram (Table 18.).

Out of the 3,252 isolates collected 1,457 isolates (44.8%) were found to be non-toxigenic clostridia. Sequencing representatives (n=181) from the non-toxigenic clostridia displayed two dominate clostridia groups Clostridium bifermentans group (Paraclostridium bifermentans and P. benzoelyticum) and Clostridium beijerinckii group (C. diolis, C. beijerinckii, C. chromiireducens, C. saccharoperbutylacetonicum, C. puniceum, and C. saccharobutylicum), the two main groups of the non-toxigenic clostridia group made up 53.6% of the non-toxigenic isolates (FIG. 68.).

Discussion:

Fecal samples were used as the most readily available sample type to estimate the level and obtain isolates of clostridia and C. perfringens within the digestive system of ruminants. From the 339 fecal samples collected throughout the Northeast region all samples had detectable levels of clostridia. The majority of the isolates harvested (55.2%) from the ruminant samples contained a toxin gene specific to C. perfringens. C. perfringens was detected in 90% of the cow fecal samples and in 70% of the calf fecal samples. The high presence of clostridia and C. perfringens indicates the risk for sub-acute enteric clostridia disease challenges in most ruminants throughout the Northeast region. C. perfringens isolates were diverse according to the RAPD genetic fingerprints but were not specific to sample type or farm. Diverse representatives of C. perfringens were mostly inhibited (>60%) by at least one bacteriocin from the following strains 747, 1104, 1541, 1781 or 2018. From the 412 isolates tested 342 isolates were inhibited by greater than 60% by at least one of the strains, representing inhibition of 84.6% of the total C. perfringens population based on representation from clusters on the RAPD dendrogram. This indicates the Bacillus strains 747, 1104, 1541, 1781 and 2018 can inhibit a wide range of diversity of C. perfringens isolates. The Bacillus strains are not limited to specific clostridia strain(s) like a vaccine which may be missing large groups of the clostridia populations based on the genetic diversity observed in the RAPD dendrogram. DNA sequencing of the non-toxigenic clostridia revealed two major identifications of Clostridium species. C. bifermentans group, which is known to produce 1,3-propanediol (Leja et al., 2014; Myszka et al., 2012) and C. beijerinckii group known to produce butanol and acetone (Hou et al., 2017). The production of the metabolic end products of these species could be having an impact in the rumen, reducing performance parameters such as milk production within a dairy cow. The high inhibition level against the C. perfringens isolates in vitro indicates a potential mode of action of the Bacillus strains 747, 1104, 1541, 1781 and 2018.

The Bacillus strains offer a prophylactic effect on the clostridia populations which may not only increase rumen efficiency leading to increased milk production, but prevent acute levels of C. perfringens reducing the occurrence of digestive deaths. The high prevalence of clostridia and C. perfringens in fecal samples collected suggests efficiency improvement opportunities in many ruminants throughout the Northeast region. This example displays the diversity of clostridia isolates from the ruminant fecal and feed samples collected from the Northeast region. The Bacillus strains tested 747, 1104, 1541, 1781 and 2018, could inhibit most of the clostridia diversity observed in the Northeast region. The product, in accordance with this embodiment of the present invention could inhibit toxigenic clostridia isolated from ruminants in the Northeast indicating a benefit in rumen efficiency if fed to dairy cows as a direct fed microbial (DFM).

TABLE 17 Fecal samples, 339, were collected from Northeast regional dairies separated by farm (9) and age (cow or calf) which were enumerated for clostridia, tested for C. perfringens, isolates were genotyped and tested for inhibition. Farm Name State Cow Fecal Calf Fecal Farm MM Maine 46 7 Farm N Vermont 63 10 Farm A Vermont 29 3 Farm BH New York 35 3 Farm D New York 34 5 Farm H New York 20 3 Farm K Vermont 48 5 Farm O New York 13 1 Farm V New York 13 1 Total 301 38

TABLE 18 Bacteriocin assay results displaying each isolate tested and the source of that isolate, isolated from Northeast regional fecal samples. Inhibition was calculated based on the percent of growth for each treatment well compared to the positive control. Sample Bacillus Strains Isolate ID Farm Type Group 747 1104 1541 1781 2018 R10.1.8 Farm HE Cow Fecal Low Cows 51.3% 0.0% 0.0% 55.3% 3.7% R10.12.9 Farm HE Cow Fecal UpClose Cows 96.2% 95.2% 94.7% 94.3% 95.4% R10.13.1 Farm HE Cow Fecal UpClose Cows 96.3% 95.7% 96.4% 96.0% 96.2% R10.13.3 Farm HE Cow Fecal UpClose Cows 96.6% 96.0% 5.5% 96.2% 96.0% R10.13.5 Farm HE Cow Fecal UpClose Cows 96.6% 95.4% 95.4% 95.0% 95.8% R10.13.6 Farm HE Cow Fecal UpClose Cows 96.6% 96.2% 97.0% 96.5% 96.5% R10.15.7 Farm HE Cow Fecal UpClose Cows 96.9% 96.0% 97.1% 96.5% 96.5% R10.16.2 Farm HE Cow Fecal Far Off (Dry) 96.6% 95.8% 96.3% 96.1% 96.2% R10.18.6 Farm HE Cow Fecal Far Off (Dry) 96.1% 95.6% 96.4% 95.7% 95.9% R10.19.4 Farm HE Cow Fecal Far Off (Dry) 93.3% 91.9% 91.7% 90.9% 91.7% R10.2.7 Farm HE Cow Fecal Far Off (Dry) 96.3% 96.0% 96.9% 96.5% 96.4% R10.21.5 Farm HE Calf Fecal Calf 95.2% 94.2% 95.1% 93.5% 94.8% R10.22.1 Farm HE Calf Fecal Calf 67.7% 25.2% 0.0% 56.8% 0.0% R10.22.3 Farm HE Calf Fecal Calf 68.8% 39.4% 0.0% 67.1% 0.0% R10.22.4 Farm HE Calf Fecal Calf 93.1% 85.1% 68.1% 91.3% 72.4% R10.23.10 Farm HE Calf Fecal Calf 97.1% 96.4% 96.5% 95.8% 96.5% R10.23.2 Farm HE Calf Fecal Calf 8.7% 0.0% 9.8% 22.7% 21.1% R10.23.3 Farm HE Calf Fecal Calf 94.8% 94.2% 88.6% 0.0% 11.2% R10.23.5 Farm HE Calf Fecal Calf 97.0% 96.2% 96.4% 96.0% 96.6% R10.23.7 Farm HE Calf Fecal Calf 95.1% 94.3% 95.2% 93.9% 93.7% R10.8.4 Farm HE Cow Fecal Low Cows 97.3% 96.8% 97.7% 96.4% 96.9% R10.9.6 Farm HE Cow Fecal Low Cows 92.3% 91.5% 93.1% 92.5% 0.0% R11.1.5 Farm BH Cow Fecal High Cows 94.5% 60.4% 94.5% 94.5% 95.6% R11.11.1 Farm BH Cow Fecal Low Cows 97.2% 96.5% 97.4% 97.1% 96.8% R11.11.2 Farm BH Cow Fecal Low Cows 92.6% 91.4% 93.4% 92.6% 92.6% R11.12.7 Farm BH Cow Fecal Low Cows 69.9% 65.0% 65.6% 0.0% 66.3% R11.14.4 Farm BH Cow Fecal Low Cows 98.6% 95.8% 97.7% 94.4% 98.1% R11.15.1. Farm BH Cow Fecal Low Cows 98.4% 84.7% 96.0% 94.8% 98.8% R11.15.7 Farm BH Cow Fecal Low Cows 99.8% 97.7% 99.6% 99.8% 99.5% R11.15.8 Farm BH Cow Fecal Low Cows 57.0% 9.5% 17.0% 9.8% 49.3% R11.16.10 Farm BH Cow Fecal Low Cows 96.9% 96.5% 97.3% 96.5% 96.7% R11.16.4 Farm BH Cow Fecal Low Cows 90.0% 46.5% 69.5% 65.0% 58.6% R11.16.5 Farm BH Cow Fecal Low Cows 37.9% 0.0% 0.0% 62.6% 0.0% R11.16.7 Farm BH Cow Fecal Low Cows 99.6% 99.6% 99.6% 99.8% 99.2% R11.16.8 Farm BH Cow Fecal Low Cows 99.6% 99.6% 99.6% 96.6% 0.0% R11.17.1 Farm BH Cow Fecal Low Cows 99.4% 56.3% 36.1% 91.4% 78.7% R11.17.3 Farm BH Cow Fecal Low Cows 96.2% 75.0% 90.4% 94.2% 92.3% R11.17.4 Farm BH Cow Fecal Low Cows 98.6% 4.9% 0.0% 86.6% 11.3% R11.17.6 Farm BH Cow Fecal Low Cows 97.1% 96.1% 96.8% 96.5% 97.2% R11.17.8 Farm BH Cow Fecal Low Cows 99.6% 0.0% 36.4% 99.3% 0.0% R11.18.10 Farm BH Cow Fecal Low Cows 96.9% 96.3% 97.9% 97.2% 96.9% R11.18.3 Farm BH Cow Fecal Low Cows 97.9% 97.4% 97.8% 97.9% 97.6% R11.18.4 Farm BH Cow Fecal Low Cows 84.2% 83.2% 86.0% 84.6% 86.0% R11.18.6 Farm BH Cow Fecal Low Cows 99.7% 0.0% 99.7% 78.7% 23.6% R11.19.1 Farm BH Cow Fecal Low Cows 93.7% 91.1% 93.8% 94.0% 93.4% R11.2.8 Farm BH Cow Fecal High Cows 95.8% 95.1% 96.0% 94.7% 90.9% R11.2.9 Farm BH Cow Fecal High Cows 96.8% 96.1% 96.5% 95.4% 96.6% R11.20.3 Farm BH Cow Fecal Low Cows 96.0% 95.3% 97.3% 96.8% 96.8% R11.20.4 Farm BH Cow Fecal Low Cows 0.0% 0.0% 0.0% 0.0% 0.0% R11.22.1 Farm BH Cow Fecal Pre-Fresh 0.0% 0.0% 0.0% 0.0% 0.0% R11.22.2 Farm BH Cow Fecal Pre-Fresh 99.6% 99.2% 98.8% 99.2% 98.8% R11.22.3 Farm BH Cow Fecal Pre-Fresh 24.5% 0.4% 12.1% 0.0% 10.1% R11.22.6 Farm BH Cow Fecal Pre-Fresh 91.7% 89.5% 88.8% 91.7% 92.8% R11.22.8 Farm BH Cow Fecal Pre-Fresh 92.0% 89.2% 74.5% 90.7% 81.3% R11.23.10 Farm BH Cow Fecal Pre-Fresh 0.0% 0.0% 0.0% 0.0% 0.0% R11.23.2 Farm BH Cow Fecal Pre-Fresh 98.6% 95.8% 97.7% 94.4% 98.1% R11.23.2 Farm BH Cow Fecal Pre-Fresh 99.3% 96.5% 99.1% 98.8% 98.8% R11.23.4 Farm BH Cow Fecal Pre-Fresh 97.5% 96.7% 98.1% 97.9% 97.1% R11.23.6 Farm BH Cow Fecal Pre-Fresh 99.7% 99.5% 99.6% 99.9% 99.5% R11.24.1 Farm BH Cow Fecal Pre-Fresh 97.1% 96.1% 96.3% 27.9% 95.9% R11.24.7 Farm BH Cow Fecal Pre-Fresh 0.0% 0.0% 0.0% 0.0% 0.0% R11.25.10 Farm BH Cow Fecal Pre-Fresh 96.0% 94.8% 94.3% 93.5% 93.9% R11.25.4 Farm BH Cow Fecal Pre-Fresh 0.0% 20.2% 0.0% 0.0% 0.0% R11.25.5 Farm BH Cow Fecal Pre-Fresh 94.5% 24.3% 53.4% 90.1% 40.4% R11.25.7 Farm BH Cow Fecal Pre-Fresh 0.0% 0.0% 0.0% 0.0% 0.0% R11.26.10 Farm BH Cow Fecal Dry Cows/ 98.3% 97.4% 98.7% 98.3% 98.0% Preg Heifers R11.26.4 Farm BH Cow Fecal Dry Cows/ 28.8% 16.4% 31.9% 28.8% 40.3% Preg Heifers R11.26.6 Farm BH Cow Fecal Dry Cows/ 0.0% 0.0% 0.0% 0.0% 0.0% Preg Heifers R11.27.1 Farm BH Cow Fecal Dry Cows/ 100.0% 96.2% 100.0% 100.0% 100.0% Preg Heifers R11.27.10 Farm BH Cow Fecal Dry Cows/ 11.5% 0.0% 0.0% 0.0% 14.9% Preg Heifers R11.27.2 Farm BH Cow Fecal Dry Cows/ 96.9% 97.1% 98.2% 97.8% 97.1% Preg Heifers R11.27.3 Farm BH Cow Fecal Dry Cows/ 78.0% 70.7% 86.6% 81.7% 84.1% Preg Heifers R11.28.3 Farm BH Cow Fecal Dry Cows/ 97.2% 96.8% 98.1% 97.4% 97.2% Preg Heifers R11.28.5 Farm BH Cow Fecal Dry Cows/ 97.5% 96.3% 98.5% 98.4% 96.3% Preg Heifers R11.3.3 Farm BH Cow Fecal High Cows 96.2% 95.2% 95.8% 94.6% 95.4% R11.3.6 Farm BH Cow Fecal High Cows 99.6% 99.2% 99.5% 99.7% 99.6% R11.3.9 Farm BH Cow Fecal High Cows 97.4% 96.3% 96.5% 96.0% 96.5% R11.33.10 Farm BH Cow Fecal Fresh Cows 79.6% 96.3% 100.0% 93.5% 82.4% R11.34.3 Farm BH Cow Fecal Fresh Cows 95.2% 69.1% 66.1% 65.0% 78.5% R11.34.8 Farm BH Cow Fecal Fresh Cows 92.8% 82.1% 91.8% 93.7% 96.9% R11.35.3 Farm BH Cow Fecal Fresh Cows 32.8% 0.0% 14.3% 0.0% 9.7% R11.35.4 Farm BH Cow Fecal Fresh Cows 96.0% 96.0% 96.9% 95.0% 95.1% R11.35.6 Farm BH Cow Fecal Fresh Cows 97.1% 95.9% 96.3% 95.1% 94.7% R11.35.9 Farm BH Cow Fecal Fresh Cows 98.9% 35.5% 97.8% 98.9% 98.2% R11.37.1 Farm BH Calf Fecal Calf 14.6% 0.8% 0.0% 1.1% 0.0% R11.37.7 Farm BH Calf Fecal Calf 72.9% 63.6% 21.5% 55.1% 57.0% R11.38.5 Farm BH Calf Fecal Calf 87.5% 0.0% 53.0% 34.5% 0.0% R11.4.3 Farm BH Cow Fecal High Cows 98.4% 84.7% 96.0% 94.8% 98.8% R11.5.2 Farm BH Cow Fecal High Cows 0.0% 0.0% 0.0% 0.0% 0.0% R11.5.7 Farm BH Cow Fecal High Cows 97.5% 96.5% 96.8% 96.2% 97.1% R11.7.5 Farm BH Cow Fecal High Cows 0.0% 0.0% 0.0% 0.0% 0.0% R11.7.8 Farm BH Cow Fecal High Cows 97.5% 96.8% 97.6% 97.4% 97.4% R11.8.5 Farm BH Cow Fecal High Cows 96.7% 96.0% 96.9% 96.9% 96.4% R11.9.6 Farm BH Cow Fecal High Cows 97.0% 96.5% 97.6% 97.4% 97.1% R14.1.10 Farm D Calf Fecal Calf 40.8% 14.3% 14.5% 49.9% 43.6% R14.1.9 Farm D Calf Fecal Calf 88.9% 2.8% 0.0% 95.0% 4.3% R14.10.10 Farm D Cow Fecal Far Dry 100.0% 26.3% 71.0% 100.0% 0.0% R14.10.7 Farm D Cow Fecal Far Dry 88.3% 85.9% 88.3% 97.0% 96.6% R14.10.8 Farm D Cow Fecal Far Dry 100.0% 100.0% 100.0% 100.0% 100.0% R14.11.2 Farm D Cow Fecal Far Dry 26.3% 0.0% 0.0% 0.0% 46.7% R14.11.6 Farm D Cow Fecal Far Dry 100.0% 100.0% 100.0% 100.0% 100.0% R14.13.4 Farm D Cow Fecal Close Dry 96.3% 45.7% 89.5% 89.1% 0.0% R14.14.9 Farm D Cow Fecal Close Dry 0.0% 0.0% 0.0% 0.0% 0.0% R14.15.10 Farm D Cow Fecal Low 0.0% 0.0% 0.0% 0.0% 0.0% R14.16.1 Farm D Cow Fecal Low 83.1% 32.8% 44.4% 50.8% 49.2% R14.16.2 Farm D Cow Fecal Low 0.0% 0.0% 0.0% 0.0% 0.0% R14.16.5 Farm D Cow Fecal Low 11.8% 0.0% 8.5% 23.1% 1.1% R14.17.1 Farm D Cow Fecal Low 97.0% 96.3% 96.5% 97.4% 95.8% R14.17.9 Farm D Cow Fecal Low 97.0% 96.0% 97.1% 96.6% 96.1% R14.18.5 Farm D Cow Fecal Low 93.6% 91.4% 93.6% 93.8% 91.9% R14.2.6 Farm D Calf Fecal Calf 95.2% 9.7% 0.0% 94.1% 20.4% R14.2.7 Farm D Calf Fecal Calf 96.2% 95.1% 96.2% 96.6% 96.6% R14.20.5 Farm D Cow Fecal High 97.3% 96.9% 97.3% 97.6% 96.4% R14.20.7 Farm D Cow Fecal High 95.9% 95.0% 95.6% 95.8% 95.1% R14.21.10 Farm D Cow Fecal High 97.1% 96.1% 97.2% 96.6% 96.3% R14.21.5 Farm D Cow Fecal High 97.2% 95.8% 97.3% 96.6% 96.2% R14.21.7 Farm D Cow Fecal High 66.3% 67.9% 75.1% 67.2% 67.0% R14.25.9 Farm D Cow Fecal High 94.4% 93.1% 96.0% 94.6% 94.2% R14.27.9 Farm D Cow Fecal High 97.2% 96.4% 98.0% 97.0% 97.0% R14.28.4 Farm D Cow Fecal High 97.0% 95.9% 97.4% 97.0% 96.4% R14.29.6 Farm D Cow Fecal High 81.8% 22.0% 36.8% 35.5% 45.6% R14.29.9 Farm D Cow Fecal High 0.0% 0.0% 0.0% 0.0% 0.0% R14.3.10 Farm D Calf Fecal Calf 97.7% 96.9% 97.8% 97.8% 97.1% R14.3.2 Farm D Calf Fecal Calf 91.7% 94.9% 93.3% 93.3% 94.2% R14.3.7 Farm D Calf Fecal Calf 96.3% 96.6% 97.6% 98.4% 96.0% R14.31.3 Farm D Cow Fecal Fresh 97.0% 95.8% 97.2% 96.6% 96.5% R14.31.8 Farm D Cow Fecal Fresh 71.2% 4.4% 2.9% 15.3% 10.9% R14.32.3 Farm D Cow Fecal Fresh 95.9% 95.3% 97.0% 96.1% 96.2% R14.39.7 Farm D Cow Fecal Fresh 97.6% 98.2% 97.6% 89.2% 97.6% R14.4.3 Farm D Calf Fecal Calf 97.7% 97.8% 97.7% 97.3% 97.1% R14.4.7 Farm D Calf Fecal Calf 97.2% 99.3% 99.4% 96.3% 96.9% R14.5.1 Farm D Calf Fecal Calf 0.0% 78.7% 99.3% 99.2% 68.9% R14.5.7 Farm D Calf Fecal Calf 83.2% 83.3% 82.8% 88.8% 97.3% R14.7.1 Farm D Cow Fecal Post Fresh 97.2% 96.8% 97.9% 96.9% 97.0% R14.8.10 Farm D Cow Fecal Far Dry 0.0% 0.0% 0.0% 0.0% 0.0% R14.8.7 Farm D Cow Fecal Far Dry 4.2% 17.1% 0.0% 20.7% 0.0% R14.9.9 Farm D Cow Fecal Far Dry 80.1% 0.0% 0.0% 58.7% 0.0% R16.10.5 Farm V Cow Fecal Milk 96.0% 96.0% 95.9% 96.5% 91.2% R16.10.8 Farm V Cow Fecal Milk 96.1% 96.1% 96.7% 96.8% 96.6% R16.10.9 Farm V Cow Fecal Milk 96.5% 96.8% 97.6% 97.4% 97.1% R16.11.3 Farm V Cow Fecal Milk 96.9% 26.3% 12.3% 57.6% 53.1% R16.11.7 Farm V Cow Fecal Milk 36.6% 11.9% 87.0% 24.6% 10.3% R16.12.8 Farm V Cow Fecal Milk 96.9% 97.0% 97.8% 98.5% 97.7% R16.13.6 Farm V Cow Fecal Milk 69.7% 89.9% 94.9% 42.9% 45.1% R16.13.9 Farm V Cow Fecal Milk 90.6% 90.9% 90.9% 93.5% 92.5% R16.3.7 Farm V Cow Fecal Dry 94.0% 94.0% 0.0% 94.9% 94.7% R16.5.3 Farm V Cow Fecal Milk 85.0% 0.0% 0.0% 0.0% 0.0% R16.9.4 Farm V Cow Fecal Milk 100.0% 96.2% 100.0% 100.0% 100.0% R20.1.1 Farm K Calf Fecal Calf 96.5% 95.0% 92.9% 92.7% 89.4% R20.1.2 Farm K Calf Fecal Calf 96.6% 93.0% 94.1% 90.8% 90.9% R20.11.6 Farm K Cow Fecal Group 1 - High 99.3% 98.5% 99.1% 99.1% 99.0% R20.13.7 Farm K Cow Fecal Group 1 - High 99.2% 98.9% 99.3% 99.2% 99.0% R20.14.8 Farm K Cow Fecal Group 1 - High 3.8% 0.0% 0.0% 0.0% 0.0% R20.15.1 Farm K Cow Fecal Group 1 - High 94.4% 90.9% 92.5% 90.8% 89.0% R20.16.6 Farm K Cow Fecal Group 2- Med 20.2% 7.8% 8.4% 9.6% 7.9% R20.17.1 Farm K Cow Fecal Group 2- Med 0.2% 0.0% 0.0% 0.0% 0.0% R20.17.2 Farm K Cow Fecal Group 2- Med 69.4% 53.3% 57.8% 43.9% 46.5% R20.17.6 Farm K Cow Fecal Group 2- Med 0.8% 0.0% 0.0% 0.0% 0.0% R20.17.7 Farm K Cow Fecal Group 2- Med 0.0% 1.8% 0.0% 5.7% 0.0% R20.17.8 Farm K Cow Fecal Group 2- Med 4.8% 19.4% 0.0% 48.9% 0.0% R20.18.1 Farm K Cow Fecal Group 2- Med 0.0% 0.0% 0.0% 0.0% 0.0% R20.18.4 Farm K Cow Fecal Group 2- Med 98.9% 98.7% 99.1% 99.1% 98.9% R20.19.2 Farm K Cow Fecal Group 2- Med 0.0% 88.7% 0.0% 0.0% 90.1% R20.19.5 Farm K Cow Fecal Group 2- Med 0.0% 0.0% 98.0% 99.0% 97.4% R20.19.7 Farm K Cow Fecal Group 2 - Med 99.4% 99.2% 99.3% 99.4% 99.2% R20.19.9 Farm K Cow Fecal Group 2- Med 3.1% 0.0% 0.0% 0.0% 0.0% R20.2.10 Farm K Calf Fecal Calf 96.5% 95.3% 31.4% 92.5% 92.0% R20.2.2 Farm K Calf Fecal Calf 99.3% 98.9% 99.0% 99.3% 98.9% R20.2.3 Farm K Calf Fecal Calf 99.5% 99.5% 99.7% 99.7% 99.5% R20.2.4 Farm K Calf Fecal Calf 96.7% 95.7% 96.3% 94.7% 90.4% R20.2.5 Farm K Calf Fecal Calf 97.0% 95.2% 96.2% 94.9% 95.0% R20.2.7 Farm K Calf Fecal Calf 96.9% 96.0% 96.7% 94.6% 94.5% R20.2.8 Farm K Calf Fecal Calf 96.0% 94.4% 92.0% 91.4% 91.5% R20.2.9 Farm K Calf Fecal Calf 96.5% 94.3% 93.4% 92.2% 88.9% R20.20.4 Farm K Cow Fecal Group 2- Med 34.8% 12.5% 0.0% 4.9% 13.7% R20.20.5 Farm K Cow Fecal Group 2- Med 88.0% 37.5% 7.7% 92.8% 0.0% R20.21.3 Farm K Cow Fecal Group 2- Med 61.5% 40.8% 0.0% 69.5% 0.0% R20.22.6 Farm K Cow Fecal Group 2- Med 100.3% 100.1% 99.9% 100.1% 100.1% R20.23.1 Farm K Cow Fecal Group 2- Med 100.3% 3.4% 98.3% 100.1% 99.9% R20.24.4 Farm K Cow Fecal Group 2- Med 0.0% 0.0% 0.0% 0.0% 0.0% R20.24.8 Farm K Cow Fecal Group 2- Med 0.0% 0.0% 0.0% 0.0% 0.5% R20.27.1 Farm K Cow Fecal Group 3- Low 27.4% 13.4% 0.0% 0.0% 0.0% R20.27.5 Farm K Cow Fecal Group 3- Low 99.0% 98.9% 99.0% 99.2% 98.9% R20.28.7 Farm K Cow Fecal Group 3- Low 1.6% 0.0% 3.4% 2.5% 0.9% R20.29.4 Farm K Cow Fecal Group 3- Low 0.0% 0.0% 0.0% 88.5% 0.0% R20.3.10 Farm K Calf Fecal Calf 0.0% 0.0% 0.0% 0.0% 0.0% R20.3.2 Farm K Calf Fecal Calf 95.7% 94.3% 91.5% 91.4% 91.2% R20.3.4 Farm K Calf Fecal Calf 25.9% 0.0% 0.0% 57.2% 24.4% R20.3.6 Farm K Calf Fecal Calf 96.7% 93.6% 94.4% 91.8% 91.9% R20.3.7 Farm K Calf Fecal Calf 95.6% 94.0% 93.3% 92.6% 93.4% R20.3.8 Farm K Calf Fecal Calf 95.8% 93.8% 92.2% 91.4% 91.4% R20.3.9 Farm K Calf Fecal Calf 97.1% 96.1% 96.0% 0.0% 95.3% R20.30.3 Farm K Cow Fecal Group 3- Low 82.0% 0.0% 0.0% 90.8% 3.4% R20.32.1 Farm K Cow Fecal Group 3- Low 23.4% 5.8% 2.3% 88.0% 2.0% R20.32.2 Farm K Cow Fecal Group 3- Low 97.7% 30.1% 47.2% 98.4% 47.4% R20.32.3 Farm K Cow Fecal Group 3- Low 30.1% 0.0% 12.3% 0.0% 0.6% R20.33.10 Farm K Cow Fecal Group 3- Low 94.0% 0.0% 6.7% 90.2% 0.0% R20.33.5 Farm K Cow Fecal Group 3- Low 4.2% 1.2% 0.9% 0.0% 4.5% R20.33.8 Farm K Cow Fecal Group 3- Low 0.0% 0.0% 0.0% 0.0% 0.0% R20.36.8 Farm K Cow Fecal Group 4- 1st 4.6% 2.0% 0.7% 0.0% 0.6% Calf R20.39.5 Farm K Cow Fecal Group 4- 1st 94.8% 93.1% 14.7% 89.8% 90.3% Calf R20.4.1 Farm K Calf Fecal Calf 97.6% 0.0% 91.3% 0.0% 8.1% R20.4.10 Farm K Calf Fecal Calf 32.5% 18.9% 5.8% 68.3% 36.2% R20.4.2 Farm K Calf Fecal Calf 65.4% 28.4% 6.0% 64.2% 37.2% R20.4.3 Farm K Calf Fecal Calf 66.7% 94.6% 12.7% 92.0% 28.3% R20.4.5 Farm K Calf Fecal Calf 97.0% 58.5% 93.5% 92.5% 43.4% R20.4.9 Farm K Calf Fecal Calf 96.6% 0.0% 70.8% 0.0% 93.3% R20.41.3 Farm K Cow Fecal Group 4- 1st 99.1% 23.6% 98.9% 99.5% 99.2% Calf R20.42.1 Farm K Cow Fecal Group 4- 1st 0.0% 15.5% 98.6% 21.9% 98.1% Calf R20.42.10 Farm K Cow Fecal Group 4- 1st 0.0% 0.0% 0.0% 0.0% 0.0% Calf R20.44.2 Farm K Cow Fecal Group 4- 1st 95.9% 91.4% 88.9% 0.0% 13.2% Calf R20.45.6 Farm K Cow Fecal Group 4- 1st 96.4% 91.6% 92.6% 90.5% 0.0% Calf R20.46.2 Farm K Cow Fecal Post Fresh 0.0% 91.0% 91.4% 90.8% 55.8% R20.46.3 Farm K Cow Fecal Post Fresh 96.6% 91.3% 91.7% 46.9% 87.8% R20.47.2 Farm K Cow Fecal Post Fresh 25.3% 85.8% 24.5% 0.0% 7.4% R20.47.5 Farm K Cow Fecal Post Fresh 94.9% 92.8% 91.7% 21.2% 66.1% R20.5.2 Farm K Calf Fecal Calf 96.2% 59.2% 29.8% 91.3% 2.4% R20.5.3 Farm K Calf Fecal Calf 97.6% 75.3% 32.5% 92.0% 37.8% R20.5.4 Farm K Calf Fecal Calf 95.3% 25.6% 22.4% 51.6% 68.8% R20.53.3 Farm K Cow Fecal Dry 82.8% 0.0% 30.5% 82.4% 22.8% R20.53.7 Farm K Cow Fecal Dry 0.0% 0.0% 98.0% 0.0% 97.5% R20.6.3 Farm K Cow Fecal Dry 15.0% 0.0% 0.0% 0.0% 0.0% R20.6.5 Farm K Cow Fecal Dry 12.4% 8.4% 2.1% 10.3% 4.4% R20.6.6 Farm K Cow Fecal Group 1 - High 96.5% 93.4% 93.7% 91.4% 6.5% R20.6.7 Farm K Cow Fecal Group 1 - High 99.9% 99.4% 99.8% 99.9% 99.5% R20.6.9 Farm K Cow Fecal Group 1 - High 99.1% 98.8% 99.3% 99.1% 99.1% R20.8.8 Farm K Cow Fecal Group 1 - High 96.3% 94.5% 94.9% 37.3% 0.0% R20.9.3 Farm K Cow Fecal Group 1 - High 45.6% 4.4% 98.2% 28.7% 31.8% R20.9.6 Farm K Cow Fecal Group 1 - High 15.9% 0.0% 4.5% 0.6% 2.3% R20.9.7 Farm K Cow Fecal Group 1 - High 98.0% 98.4% 98.4% 98.7% 97.7% R21.1.1 Farm A Calf Fecal Calf 29.6% 46.2% 19.0% 52.8% 72.7% R21.1.2 Farm A Calf Fecal Calf 96.4% 93.1% 14.4% 91.3% 73.8% R21.1.3 Farm A Calf Fecal Calf 8.9% 0.0% 0.0% 18.1% 55.2% R21.1.4 Farm A Calf Fecal Calf 94.3% 94.0% 2.6% 6.2% 2.0% R21.1.8 Farm A Calf Fecal Calf 61.8% 12.4% 12.4% 85.9% 80.8% R21.10.2 Farm A Cow Fecal Group 1 98.5% 95.7% 99.8% 99.8% 78.4% R21.12.2 Farm A Cow Fecal Group 1 8.3% 0.0% 97.4% 103.6% 103.2% R21.14.1 Farm A Cow Fecal Group 2 92.4% 93.8% 99.5% 95.3% 93.4% R21.16.4 Farm A Cow Fecal Group 2 100.6% 100.6% 100.1% 100.4% 100.1% R21.18.2 Farm A Cow Fecal Group 2 98.7% 98.7% 99.8% 99.0% 99.0% R21.2.2 Farm A Calf Fecal Calf 97.0% 96.1% 94.4% 94.0% 92.2% R21.2.3 Farm A Calf Fecal Calf 53.7% 21.1% 26.7% 81.9% 33.3% R21.2.5 Farm A Calf Fecal Calf 98.1% 29.2% 97.5% 98.6% 98.1% R21.2.6 Farm A Calf Fecal Calf 72.8% 44.9% 0.0% 82.4% 25.3% R21.2.8 Farm A Calf Fecal Calf 39.1% 22.8% 22.2% 54.5% 31.7% R21.2.9 Farm A Calf Fecal Calf 99.6% 99.5% 99.6% 99.6% 99.5% R21.22.4 Farm A Cow Fecal Group 2 0.0% 0.0% 0.0% 0.0% 0.0% R21.22.6 Farm A Cow Fecal Group 2 94.7% 90.0% 90.8% 87.5% 88.2% R21.23.2 Farm A Cow Fecal Pre-Fresh 0.0% 0.0% 0.0% 0.0% 0.0% R21.23.7 Farm A Cow Fecal Pre-Fresh 2.4% 0.0% 0.0% 0.0% 0.0% R21.25.8 Farm A Cow Fecal Pre-Fresh 100.2% 100.0% 99.8% 100.0% 100.0% R21.26.10 Farm A Cow Fecal Pre-Fresh 89.4% 85.0% 82.8% 79.7% 0.0% R21.26.2 Farm A Cow Fecal Pre-Fresh 0.0% 0.0% 0.5% 3.5% 0.0% R21.26.3 Farm A Cow Fecal Pre-Fresh 100.1% 100.1% 100.0% 100.3% 100.1% R21.26.5 Farm A Cow Fecal Pre-Fresh 101.3% 40.0% 60.0% 101.3% 96.3% R21.26.7 Farm A Cow Fecal Pre-Fresh 95.4% 86.8% 92.7% 95.7% 94.9% R21.29.1 Farm A Cow Fecal Dry 92.6% 35.6% 88.8% 80.8% 0.0% R21.3.1 Farm A Calf Fecal Calf 95.8% 93.0% 0.0% 91.4% 91.1% R21.3.2 Farm A Calf Fecal Calf 95.5% 90.5% 91.5% 90.8% 77.7% R21.30.8 Farm A Cow Fecal Dry 94.7% 91.6% 90.2% 88.2% 87.6% R21.31.2 Farm A Cow Fecal Dry 95.1% 93.8% 92.0% 91.8% 89.6% R21.31.3 Farm A Cow Fecal Dry 91.9% 89.2% 0.0% 84.6% 84.2% R21.5.8 Farm A Cow Fecal Group 1 96.3% 92.9% 4.3% 91.4% 2.8% R21.6.1 Farm A Cow Fecal Group 1 81.4% 18.2% 0.0% 2.8% 0.0% R21.7.1 Farm A Cow Fecal Group 1 6.8% 0.0% 0.0% 0.0% 0.0% R21.8.3 Farm A Cow Fecal Group 1 88.8% 0.0% 78.0% 70.6% 71.3% R21.8.6 Farm A Cow Fecal Group 1 96.5% 30.2% 0.3% 73.3% 77.6% R46.14.1 Farm N Cow Fecal Pre-Fresh 0.0% 0.0% 0.0% 0.0% 0.0% R46.16.5 Farm N Cow Fecal Pre-Fresh 99.7% 98.3% 99.4% 99.4% 99.7% R46.16.9 Farm N Cow Fecal Pre-Fresh 99.8% 99.8% 99.7% 99.7% 99.7% R46.17.6 Farm N Cow Fecal Pre-Fresh 99.6% 99.6% 99.4% 99.6% 99.6% R46.19.3 Farm N Cow Fecal Pre-Fresh 99.4% 99.6% 99.4% 99.4% 99.6% R46.20.1 Farm N Cow Fecal Pre-Fresh 98.4% 97.7% 97.9% 98.4% 98.2% R46.22.4 Farm N Cow Fecal Lactating Fresh 99.7% 93.5% 99.4% 99.4% 99.6% R46.23.3 Farm N Cow Fecal Lactating Fresh 99.7% 99.9% 99.3% 99.6% 99.6% R46.23.5 Farm N Cow Fecal Lactating Fresh 100.0% 0.0% 98.8% 98.8% 101.3% R46.25.10 Farm N Cow Fecal Lactating Fresh 96.7% 95.5% 95.4% 98.2% 95.3% R46.25.6 Farm N Cow Fecal Lactating Fresh 100.0% 100.0% 99.8% 100.0% 99.8% R46.26.6 Farm N Cow Fecal Lactating Fresh 99.5% 98.9% 98.9% 99.5% 99.3% R46.27.5 Farm N Cow Fecal Lactating Fresh 99.9% 100.0% 100.0% 100.0% 99.9% R46.27.6 Farm N Cow Fecal Lactating Fresh 0.0% 0.0% 0.0% 0.0% 0.0% R46.27.8 Farm N Cow Fecal Lactating Fresh 85.0% 75.0% 75.0% 85.0% 90.0% R46.27.9 Farm N Cow Fecal Lactating Fresh 99.8% 99.8% 99.4% 99.7% 99.7% R46.28.4 Farm N Cow Fecal Lactating Fresh 99.8% 99.0% 99.0% 99.8% 99.3% R46.29.8 Farm N Cow Fecal Lactating Fresh 88.3% 77.0% 79.4% 78.3% 80.6% R46.3.5 Farm N Calf Fecal Calf 101.1% 0.0% 97.8% 89.1% 0.0% R46.31.2 Farm N Cow Fecal Lactating Fresh 99.7% 99.7% 99.6% 99.7% 99.6% R46.31.4 Farm N Cow Fecal Lactating Fresh 90.3% 88.7% 90.3% 91.9% 91.9% R46.31.5 Farm N Cow Fecal Lactating Fresh 58.1% 42.7% 60.4% 56.7% 51.7% R46.31.6 Farm N Cow Fecal Lactating Fresh 99.4% 99.4% 99.1% 99.3% 99.3% R46.31.7 Farm N Cow Fecal Lactating Fresh 100.0% 100.0% 100.0% 100.0% 100.0% R46.31.9 Farm N Cow Fecal Lactating Fresh 100.0% 99.8% 99.5% 99.8% 99.8% R46.34.10 Farm N Cow Fecal 1st Calf 99.6% 99.3% 99.5% 99.3% 99.3% R46.35.7 Farm N Cow Fecal 1st Calf 90.1% 36.3% 50.5% 71.2% 86.8% R46.35.8 Farm N Cow Fecal 1st Calf 99.8% 98.9% 98.9% 99.7% 99.7% R46.35.9 Farm N Cow Fecal 1st Calf 98.5% 27.7% 94.8% 98.4% 59.5% R46.37.2 Farm N Cow Fecal 1st Calf 100.0% 99.7% 99.9% 100.0% 100.0% R46.40.7 Farm N Cow Fecal 1st Calf 100.0% 50.0% 100.0% 50.0% 50.0% R46.42.1 Farm N Cow Fecal Low 118.2% 100.0% 81.8% 109.1% 100.0% R46.42.4 Farm N Cow Fecal Low 97.4% 98.8% 97.8% 98.8% 94.5% R46.42.6 Farm N Cow Fecal Low 99.9% 99.5% 99.9% 100.0% 86.6% R46.42.7 Farm N Cow Fecal Low 100.1% 100.1% 100.0% 100.0% 100.0% R46.43.1 Farm N Cow Fecal Low 97.6% 100.0% 26.8% 0.0% 100.0% R46.43.3 Farm N Cow Fecal Low 100.0% 100.0% 100.0% 100.0% 100.0% R46.43.9 Farm N Cow Fecal Low 99.9% 100.0% 99.4% 100.0% 99.9% R46.44.6 Farm N Cow Fecal Low 100.0% 96.8% 99.7% 100.0% 100.0% R46.45.3 Farm N Cow Fecal Low 98.5% 100.0% 99.9% 99.9% 100.0% R46.45.5 Farm N Cow Fecal Low 88.3% 86.0% 85.4% 84.7% 91.8% R46.45.8 Farm N Cow Fecal Low 100.0% 0.0% 100.0% 58.8% 100.0% R46.46.3 Farm N Cow Fecal Low 89.1% 94.2% 100.0% 83.9% 81.8% R46.46.4 Farm N Cow Fecal Low 99.5% 99.8% 99.6% 99.5% 99.6% R46.46.7 Farm N Cow Fecal Low 100.0% 99.7% 99.9% 100.0% 99.9% R46.47.2 Farm N Cow Fecal Low 99.4% 98.9% 97.7% 100.0% 100.0% R46.47.4 Farm N Cow Fecal Low 100.0% 95.9% 100.0% 100.0% 100.0% R46.49.5 Farm N Cow Fecal Low 100.0% 69.4% 100.0% 100.0% 100.0% R46.50.10 Farm N Cow Fecal Low 98.8% 98.6% 93.8% 96.5% 97.6% R46.50.8 Farm N Cow Fecal Low 100.0% 100.0% 100.0% 100.0% 98.8% R46.51.5 Farm N Cow Fecal Low 100.0% 100.0% 99.7% 100.0% 100.0% R46.52.1 Farm N Cow Fecal High 99.8% 96.3% 99.8% 100.0% 99.8% R46.52.4 Farm N Cow Fecal High 100.0% 97.9% 99.5% 100.0% 99.9% R46.53.9 Farm N Cow Fecal High 0.0% 0.0% 0.0% 0.0% 0.0% R46.54.1 Farm N Cow Fecal High 99.0% 99.2% 99.0% 74.8% 98.6% R46.54.10 Farm N Cow Fecal High 100.0% 98.7% 100.0% 100.0% 100.0% R46.54.4 Farm N Cow Fecal High 100.0% 99.2% 99.2% 100.0% 99.2% R46.54.6 Farm N Cow Fecal High 100.0% 73.6% 100.0% 100.0% 100.0% R46.54.7 Farm N Cow Fecal High 100.0% 100.0% 100.0% 100.0% 100.0% R46.58.1 Farm N Cow Fecal High 12.4% 0.0% 94.9% 0.0% 97.1% R46.58.6 Farm N Cow Fecal High 100.0% 100.0% 100.0% 87.5% 100.0% R46.60.2 Farm N Cow Fecal High 100.0% 9.9% 59.4% 71.3% 100.0% R46.60.5 Farm N Cow Fecal High 0.0% 28.7% 0.0% 0.0% 0.0% R46.60.6 Farm N Cow Fecal High 97.7% 97.4% 96.1% 98.3% 96.4% R46.60.9 Farm N Cow Fecal High 100.0% 0.0% 100.0% 100.0% 0.0% R46.61.3 Farm N Cow Fecal High 80.6% 0.0% 0.0% 83.7% 46.0% R46.62.2 Farm N Cow Fecal High 1.3% 24.1% 31.6% 2.5% 0.0% R46.62.9 Farm N Cow Fecal High 37.1% 0.0% 0.0% 64.1% 40.3% R46.64.2 Farm N Cow Fecal Dry 100.0% 100.0% 100.0% 100.0% 100.0% R46.65.3 Farm N Cow Fecal Dry 99.8% 99.8% 99.6% 99.8% 99.6% R46.65.4 Farm N Cow Fecal Dry 96.8% 98.9% 99.3% 98.4% 98.8% R46.65.8 Farm N Cow Fecal Dry 98.5% 98.5% 99.0% 99.0% 99.5% R46.66.2 Farm N Cow Fecal Dry 100.0% 98.5% 99.8% 99.7% 99.5% R46.66.6 Farm N Cow Fecal Dry 99.9% 99.9% 99.7% 99.9% 99.9% R46.67.3 Farm N Cow Fecal Dry 98.6% 98.1% 98.0% 98.6% 98.4% R46.68.7 Farm N Cow Fecal Dry 99.9% 99.9% 99.6% 99.9% 99.9% R46.69.8 Farm N Cow Fecal Dry 98.0% 0.0% 95.7% 98.6% 96.1% R46.7.1 Farm N Calf Fecal Calf 98.2% 85.4% 89.0% 80.8% 67.8% R46.7.3 Farm N Calf Fecal Calf 91.4% 0.0% 98.9% 100.0% 100.0% R46.72.10 Farm N Cow Fecal Dry 99.4% 97.5% 98.1% 98.7% 99.3% R46.73.5 Farm N Cow Fecal Dry 100.0% 99.6% 99.6% 100.1% 99.9% R46.73.9 Farm N Cow Fecal Dry 78.2% 70.5% 80.8% 76.9% 78.2% R48.1.8 Farm MM Calf Fecal Calf 99.4% 99.4% 99.2% 99.4% 98.3% R48.16.3 Farm MM Cow Fecal Pre-Fresh 69.2% 65.7% 75.6% 59.6% 74.5% R48.18.10 Farm MM Cow Fecal Pre-Fresh 98.8% 99.6% 99.7% 99.7% 93.1% R48.18.7 Farm MM Cow Fecal Pre-Fresh 91.5% 83.9% 99.4% 95.2% 94.0% R48.19.1 Farm MM Cow Fecal Pre-Fresh 99.7% 88.7% 99.5% 99.5% 98.2% R48.19.10 Farm MM Cow Fecal Pre-Fresh 86.3% 97.1% 99.9% 89.8% 99.9% R48.19.2 Farm MM Cow Fecal Pre-Fresh 97.7% 98.9% 99.8% 93.7% 98.4% R48.19.3 Farm MM Cow Fecal Pre-Fresh 100.0% 96.6% 88.7% 100.2% 100.2% R48.19.7 Farm MM Cow Fecal Pre-Fresh 98.6% 74.8% 93.6% 94.9% 95.8% R48.21.9 Farm MM Cow Fecal Pre-Fresh 98.5% 63.1% 74.0% 99.4% 65.8% R48.22.3 Farm MM Cow Fecal Pre-Fresh 43.1% 45.0% 49.9% 43.4% 45.8% R48.24.5 Farm MM Cow Fecal Fresh 65.0% 40.7% 55.3% 53.9% 70.6% R48.24.9 Farm MM Cow Fecal Fresh 93.2% 99.7% 99.7% 94.9% 99.7% R48.26.8 Farm MM Cow Fecal Fresh 55.6% 55.4% 44.2% 56.9% 57.6% R48.28.1 Farm MM Cow Fecal Fresh 97.4% 97.1% 94.8% 71.5% 96.7% R48.28.2 Farm MM Cow Fecal Fresh 93.8% 94.0% 93.5% 94.9% 94.5% R48.30.1 Farm MM Cow Fecal Low 5.9% 5.9% 9.1% 5.4% 0.2% R48.31.9 Farm MM Cow Fecal Low 98.2% 99.6% 98.9% 98.7% 98.7% R48.35.3 Farm MM Cow Fecal Mid-Lac 99.6% 99.8% 99.5% 99.8% 99.8% R48.35.8 Farm MM Cow Fecal Mid-Lac 82.3% 98.9% 96.5% 100.0% 100.0% R48.36.4 Farm MM Cow Fecal Mid-Lac 95.6% 42.1% 99.7% 93.8% 99.8% R48.36.5 Farm MM Cow Fecal Mid-Lac 98.2% 93.6% 97.5% 83.6% 97.4% R48.38.4 Farm MM Cow Fecal Mid-Lac 75.4% 82.0% 97.9% 98.9% 97.0% R48.40.10 Farm MM Cow Fecal Mid-Lac 99.4% 28.3% 98.5% 98.9% 19.7% R48.40.2 Farm MM Cow Fecal Mid-Lac 99.4% 68.1% 98.4% 99.9% 100.0% R48.41.9 Farm MM Cow Fecal Mid-Lac 63.8% 58.1% 93.3% 58.4% 77.3% R48.44.6 Farm MM Cow Fecal Mid-Lac 100.0% 99.9% 99.3% 99.6% 100.0% R48.44.8 Farm MM Cow Fecal Mid-Lac 61.5% 19.4% 66.0% 56.4% 75.7% R48.46.8 Farm MM Cow Fecal Mid-Lac 60.1% 13.6% 78.6% 58.4% 71.4% R48.46.9 Farm MM Cow Fecal Mid-Lac 73.8% 28.4% 77.1% 75.4% 65.0% S50.4.1 Farm BH Feed Fresh Cow TMR 0.0% 0.0% 0.0% 0.0% 0.0% S50.4.6 Farm BH Feed Fresh Cow TMR 0.0% 0.0% 0.0% 0.0% 0.0% S50.5.3 Farm BH Feed Prefresh TMR 0.0% 0.0% 0.0% 0.0% 0.0% S50.5.5 Farm BH Feed Prefresh TMR 0.0% 0.0% 0.0% 0.0% 0.0% S50.5.8 Farm BH Feed Prefresh TMR 0.0% 0.0% 0.0% 10.4% 13.9% S50.6.1 Farm BH Feed High Cow TMR 0.0% 0.0% 0.0% 0.0% 0.0% S50.6.3 Farm BH Feed High Cow TMR 0.0% 0.0% 0.0% 0.0% 0.0% S51.2.2 Farm HE Feed Haylage 1.9% 0.0% 0.0% 0.0% 0.0% S54.1.1 Farm D Feed Wet Brewers Grain 89.8% 91.3% 93.1% 0.0% 2.6% S54.16.2 Farm D Feed Fresh Cow TMR 0.0% 0.0% 0.0% 0.0% 0.0% S54.18.3 Farm D Feed High Cow TMR 0.0% 0.0% 0.0% 0.0% 0.0% S54.19.4 Farm D Feed Far Off Dry TMR 0.0% 0.0% 0.0% 0.0% 0.0% S55.9.1 Farm V Feed Dry Cow TMR 30.4% 5.7% 0.0% 19.2% 0.0% S56.1.2 Farm O Feed Pellets 98.1% 96.7% 97.5% 96.6% 96.2% S56.1.3 Farm O Feed Pellets 11.8% 1.9% 5.5% 0.0% 4.5% S56.7.6 Farm O Feed Corn Silage Face 38.8% 4.5% 0.0% 13.2% 32.4% S56.9.1 Farm O Feed Milk Cow TMR 40.6% 39.7% 56.7% 39.8% 54.8% S56.9.3 Farm O Feed Milk Cow TMR 4.6% 0.0% 6.1% 0.0% 11.5% S56.9.6 Farm O Feed Milk Cow TMR 0.0% 0.0% 0.0% 0.0% 0.0% S56.9.7 Farm O Feed Milk Cow TMR 0.0% 0.0% 0.0% 0.0% 0.0% S61.10.6 Farm K Feed TMR 1 44.5% 20.2% 6.8% 54.3% 16.4% S61.12.1 Farm K Feed TMR 3 57.1% 0.0% 40.8% 12.8% 9.3% S61.12.10 Farm K Feed TMR 3 0.0% 0.0% 0.0% 0.0% 0.0% S61.12.5 Farm K Feed TMR 3 0.0% 0.0% 0.0% 0.0% 0.0% S61.12.7 Farm K Feed TMR 3 0.0% 0.0% 0.0% 0.0% 0.0% S61.3.2 Farm K Feed Corn Silage Face 95.0% 92.8% 92.3% 90.3% 89.9% S61.3.4 Farm K Feed Corn Silage Face 0.0% 94.4% 94.4% 0.0% 92.1% S61.5.3 Farm K Feed Dry Hay Ontario 95.3% 7.7% 98.3% 89.8% 95.1% S91.2.4 Farm N Feed Grass Haylage 82.4% 77.4% 80.5% 86.6% 82.2% S91.2.5 Farm N Feed Grass Haylage 77.9% 50.5% 57.2% 74.1% 40.4% S91.3.5 Farm N Feed Pre Fresh 65.5% 73.5% 76.1% 64.5% 66.7% S91.5.4 Farm N Feed Low TMR 73.8% 76.3% 74.9% 62.4% 66.0%

Example 10: Selection of Bacillus Strains to Inhibit Clostridium Perfringens and Non-Toxigenic Clostridia Isolated from Ruminant Fecal Samples. (Mid-Atlantic)

Introduction:

Clostridium is a genus of Gram-positive, spore-forming bacteria that are common residents of the gastrointestinal tract. A number of Clostridium species have been linked to enteric disease in ruminants including hemorrhagic bowel syndrome (HBS), a disease often correlated to elevated levels of Clostridium perfringens Type A. While most of the enteric diseases caused by clostridia are acute and occur sporadically in herds, in general, the prognosis is poor and the first sign of illness may be death. Based on recent results sub-acute enteric clostridia disease challenges may be a more wide spread issue than acute challenges. Due to a low success rate from treatment in acute disease challenges a more common, emphasis needs to be placed on prophylactic measures.

The purpose of this research was to characterize the distribution and diversity of clostridia in ruminants and ensure inhibition of these isolates using novel Bacillus strains as a method to control the clostridia populations.

Materials and Methods:

Fecal samples (186) from cows, heifers and calves gathered from 6 farms in the Mid-Atlantic Region were diluted 1:10 with sterile peptone, heat shocked for 30 minutes at 60° C., enumerated in sterile peptone and pour plated on Tryptose Sulphite Cycloserine (TSC) agar with D-cycloserine (400 mg/L) to select for clostridia species. Agar plates were incubated at 37° C. anaerobically for 24 hours. If present, isolated sulphite-reducing colonies were counted for a total clostridia count (CFU/g) and representative isolates were picked into Reinforced clostridia Medium (RCM) (Oxoid, CM0149) and incubated anaerobically for 24 hours at 37° C. After 24 hours of incubation the cultures were transferred (10%) to Brain Heart Infusion (BHI) broth (BD, 211059) and incubated anaerobically for 24 hours at 37° C.

DNA extractions were performed in 96-well blocks containing 500 μl presumptive clostridia culture per well. Cells were harvested by centrifugation at 4,700 rpm for 10 minutes, the supernatant was removed. Cells were re-suspended in 500 μl of 50 mM of EDTA-2Na (pH=8.0). Aliquots of 300 μl of the suspended cells were transferred to a new 96-well block and combined with 20 μl of lysozyme from chicken egg white (Sigma, L6876) solution (100 mg/ml in 50 mM EDTA) to lyse bacterial cells. The 96-well block was incubated for 1 hour at 37° C. to lyse bacterial cells. Following the incubation 220 μl of lysis buffer (6 M Guanidine, 20% Triton-X 100, 10 mM Tris-HCL, pH 7.5) was added, mixed then incubated at room temperature for 15 minutes. Following the incubation 20 μl of Proteinase K (NEB, 800 U/mL) was added to each well, mixed and incubated at 55° C. for 30 minutes to degrade proteins. The cell lysate was then transferred to 96-well binding plate (Promega, A2278) and centrifuged at 4,700 rpms for 5 minutes. Flow through was discarded, three washes of the binding plate columns were executed centrifuging 750 μl of Column Wash Solution (Promega, A1318) at 4700 rpms for 1 minute and 30 seconds discarding flow through at the end of each spin. The binding plate was centrifuged for an additional 10 minutes at 4,700 rpm to remove any residual ethanol. A clean elution plate was then placed under the binding plate and DNA was eluted with 200 μl, pre-warmed (55° C.), Nuclease Free Water (Promega, P1195).

DNA was screened for toxin genes (α, β, ε, and ι) specific to C. perfringens using polymerase chain reaction (PCR). Amplification of toxin genes was executed using a multiplex PCR containing four primer sets (Yoo et al., 1997) (Table 1.) The PCR mixture contained 2.5 μl 10×PCR Buffer, 2 μl of 50 mM MgCl₂, 0.5 μM of each primer (Table 1.), 0.1 μl of Invitrogen™ Platinum™ Taq DNA Polymerase, 2.5 μl of DNA, sterile water was added to achieve 25 μl for a total reaction volume. The mixture underwent 5 minutes at 94° C., followed by 30 cycles of 94° C. for 1 minute, 55° C. for 1 minute, 72° C. for 1 minute finishing with a final elongation of 3 minutes at 72° C. PCR products were observed using a Fragment Analyzer (Advanced Analytics) to determine if amplification was achieved. If one or multiple toxin genes were observed a toxin type identification was assigned to each isolate based on their toxin gene profile (Songer, 1996). C. perfringens positive to total clostridia isolate ratio was used to calculate an estimated C. perfringens count based on the total clostridia count.

Unique strain-specific genetic fingerprints were generated using Random Amplification of Polymorphic DNA (RAPD) analysis on select isolates to determine diversity among fecal C. perfringens isolates. The PCR contained 5 μl of DNA, 2.5 μl RAPD primer 2 (10 μM) (Table 1.), and 17.5 μl of sterile water which was added to a Ready-To-Go RAPD Analysis Bead (Life Sciences, 27-9500-01). The mixture underwent 5 minutes at 95° C., followed by 45 cycles of 95° C. for 1 minute, 36° C. for 1 minute, 72° C. for 2 minutes finishing with a final elongation of 5 minutes at 72° C. PCR products observed on a Fragment Analyzer (Advanced Analytics) to determine amplification patterns and were imported into BioNumerics, bioinformatics software, for analysis. RAPD patterns were compared with a band based Dice correlation analysis method to determine the similarity between RAPD patterns as a way to monitor diversity between isolates.

Antimicrobial screening was done on C. perfringens isolates obtained from ruminant samples to gauge the effectiveness of the antimicrobial bacteriocin produced by the inventors' identified Bacillus strains 747, 1104, 1541, 1781, and 2018. Bacteriocin was harvested by growing each strain at 32° C. in a shaking incubator at 150 rpms for 24 hours in Brain Heart Infusion (BHI) broth. A 1% transfer of the 24-hour culture to fresh BHI broth was executed after incubation. The Bacillus were then incubated for 36-48 hours in a 32° C. shaking incubator at 150 rpms. The culture was then centrifuged at 14,000×g for 20 minutes, supernatant was then filtered with a 0.2 m filter to remove any residual cells.

A bacteriocin turbidity assay was executed by growing C. perfringens strains isolated from ruminant fecal samples in RCM for 24 hours, anaerobically, at 37° C. Overnight culture was transferred (1%) to sterile RCM and immediately used in the assay. For each C. perfringens isolate at least six wells were run in a sterile 48 well reaction plate, 600 μl inoculated culture (positive control), 600 μl inoculated RCM+70 μl bacteriocin (747, 1104, 1541, 1781, and 2018) and 670 RCM (un-inoculated, negative control). Plates were incubated anaerobically at 37° C. for 24 hours then read using a BioTek Epoch Microplate Spectrophotometer, readings were taken at a wavelength of 600 nm. Optical density readings from the negative controls were subtracted from all OD readings and percent inhibition was calculated using the positive control and each bacteriocin treatment.

To identify clostridia that did not have at least one toxin gene specific to C. perfringens, a PCR reaction was performed on the isolate DNA to amplify the 16S region of rDNA using primers 27F-YM and 1492R-Y (Table 1). This was done on 20% of the isolates that did not contain a toxin gene specific to C. perfringens. The PCR mixture contained 5 μl of 10×PCR Buffer, 2 μl of 50 mM MgCl₂, 1 μl of 50 mM dNTPs, 0.4 μM of each primer (Table 1.), 0.2 μl of Invitrogen™ Platinum™ Taq DNA Polymerase, 5 μl of DNA, and sterile water was added to achieve 50 μl for a total reaction volume. The mixture underwent 4 minutes at 95° C., followed by 35 cycles of 95° C. for 30 seconds, 50° C. for 30 seconds, 72° C. for 2 minutes finishing with a final elongation of 7 minutes at 72° C. A quality check was done on the amplification and PCR product was sent to gene wiz (https://www.genewiz.com) to obtain the sequences for the 16S genes. Sequences were compared to known typed bacterial strains obtained from EZbiocloud online electronic database (http://www.ezbiocloud.net/). Based on comparisons of these sequences a bacterial identification was assigned to the isolates.

Results:

Fecal samples, 186, were collect from 6 Mid-Atlantic region (Pennsylvania and Virginia) dairy farms from which 2,026 presumptive clostridia isolates were isolated as representatives of the clostridial diversity in Mid-Atlantic dairies (Table 19.).

Clostridia enumeration results indicated the average level of clostridia CFU/g across all calf fecal samples was 58,500 CFU/g with individual fecal samples ranging from 35 to 615,000 CFU/g. While the average level of clostridia CFU/g across all cow fecal samples was 2,570 CFU/g with individual fecal samples ranging from 20 to 147,000 CFU/g (FIG. 69.). Samples appeared to range in clostridia levels for both calves (FIG. 70.) and cows (FIG. 71.).

C. perfringens enumeration results displayed the average level of C. perfringens CFU/g across all calf fecal samples was 1,100 CFU/g with individual samples ranging from <10 to 5,640 CFU/g. While the average level of C. perfringens CFU/g across all cow fecal samples was 1,430 CFU/g with individual samples ranging from <10 to 87,900 CFU/g (FIG. 72.). Samples appeared to range in clostridia levels for both calves (FIG. 73.) and cows (FIG. 74.).

Analysis of the toxin multiplex PCR results displayed which isolates contained toxin genes specific to C. perfringens. A total of 2,026 presumptive clostridia isolates from Mid Atlantic fecal samples have been tested for the indicated C. perfringens toxin genes. Of the 2,026 clostridia isolates screened, 649 isolates (32%) tested positive for at least 1 of the toxin genes. From the 649 toxin-gene positive isolates 630 (97.1%) were identified as Type A (αtoxin only), however β, ε and ι, toxins were also detected in the clostridia isolates.

Gentic RAPD fingerprint patterns displayed diversity among the 582 isolates that successful amplified. The isolates tested were harvested from calf fecal, cow fecal and feed and did not cluster strictly based on the sample type or farm. Isolates formed 85 clusters based on 75% similarity according to the Dice correlation method. The largest cluster was 55 isolates (9.5%) and had a shannon idenx of diversity of 4.07.

Representatives from the RAPD dendrogram were selected to capture the diversity of the C. perfringens population from this region and subjected to inhibition assays. Antimicrobial testing using the bacteriocin turbidity assay displayed good inhibition of most ruminant fecal C. perfringens isolates using bacteriocin harvested from 747, 1104, 1541, 1781, and 2018. The bacteriocin from at least one of the strains 747, 1104, 1541, 1781, and 2018 were able to inhibit the growth >60% of 103 of the 129 isolates tested representing a total of 85.9% inhibition of the C. perfringens population based on the dendrogram (Table 20.).

Out of the 2,026 isolates collected 1,377 isolates (68%) were found to be non-toxigenic clostridia. Sequencing representatives (n=151) from the non-toxigenic clostridia displayed one dominate clostridia groups, Clostridium bifermentans group (Paraclostridium bifermentans and P. benzoelyticum). Clostridium bifermentans made up 51.5% of the non-toxigenic isolates (FIG. 75.).

Discussion:

Fecal samples were used as the most readily available sample type to estimate the level and obtain isolates of clostridia and C. perfringens within the digestive system of ruminants. From the 186 fecal samples collected throughout the Mid-Atlantic region all samples had detectable levels of clostridia. Many isolates harvested (649 isolates) from the ruminant samples contained a toxin gene specific to C. perfringens. C. perfringens was detected in 72% of the cow fecal samples and in 50% of the calf fecal samples. The high presence of clostridia and C. perfringens indicates the risk for sub-acute enteric clostridia disease challenges in most ruminants throughout the Mid-Atlantic region. C. perfringens isolates were diverse according to the RAPD genetic fingerprints but were not specific to sample type or farm. Diverse representatives of C. perfringens were mostly inhibited (>60%) by at least one bacteriocin from the following strains 747, 1104, 1541, 1781 or 2018. From the 129 isolates tested 103 isolates were inhibited by greater than 60% by at least one of the strains, representing inhibition of 85.9% of the total C. perfringens population based on representation from clusters on the RAPD dendrogram. This indicates the Bacillus strains 747, 1104, 1541, 1781 and 2018 can inhibit a wide range of diversity of C. perfringens isolates. The Bacillus strains are not limited to specific clostridia strain(s) like a vaccine which may be missing large groups of the clostridia populations based on the genetic diversity observed in the RAPD dendrogram. DNA sequencing of the non-toxigenic clostridia revealed one major identification of Clostridium species. C. bifermentans group, which is known to produce 1,3-propanediol (Leja et al., 2014; Myszka et al., 2012). The high inhibition level against the C. perfringens isolates in vitro indicates a potential mode of action of the Bacillus strains 747, 1104, 1541, 1781 and 2018.

The Bacillus strains offer a prophylactic effect on the clostridia populations which may not only increase rumen efficiency leading to increased milk production, but prevent acute levels of C. perfringens reducing the occurrence of digestive deaths. The high prevalence of clostridia and C. perfringens in fecal samples collected suggests efficiency improvement opportunities in many ruminants throughout the Mid-Atlantic region. This example displays the diversity of clostridia isolates from the ruminant fecal and feed samples collected from the Mid-Atlantic region. The Bacillus strains tested 747, 1104, 1541, 1781 and 2018, were able to inhibit most of the clostridia diversity observed in the Mid-Atlantic region. The product, in accordance with this embodiment of the present invention was able to inhibit toxigenic clostridia isolated from ruminants in the Mid-Atlantic region indicating a benefit in rumen efficiency if fed to dairy cows as a direct fed microbial (DFM).

TABLE 19 Fecal samples, 186, were collected from Mid-Atlantic dairies separated by farm (6) and age (cow or calf) which were enumerated for clostridia, tested for C. perfringens, isolates were genotyped and tested for inhibition. Farm Name State Cow Fecal Calf Fecal Farm B PA 11 1 Farm MD PA 68 7 Farm RR PA 10 1 Farm RG PA 17 1 Farm RH VA 12 2 Farm WH PA 56 0 Total 174 12

TABLE 20 Bacteriocin assay results displaying each isolate tested and the source of that isolate, isolated from the Mid-Atlantic region fecal samples. Inhibition was calculated based on the percent of growth for each treatment well compared to the positive control. Isolate Sample Bacillus Strains ID Farm Type Group 747 1104 1541 1781 2018 R37.4.3 Farm Cow Heifer 100.0% 99.7% 99.2% 99.6% 100.0% RH Fecal R37.4.9 Farm Cow Heifer 100.0% 99.8% 99.4% 99.4% 100.0% RH Fecal R37.4.10 Farm Cow Heifer 100.0% 99.6% 99.6% 99.0% 99.8% RH Fecal R37.5.1 Farm Cow Lactating Cow 95.8% 90.4% 89.8% 93.1% 94.3% RH Fecal R37.7.5 Farm Cow Lactating Cow 99.8% 99.6% 99.8% 98.3% 99.9% RH Fecal R37.10.3 Farm Cow Lactating Cow 99.7% 99.7% 99.7% 98.5% 99.7% RH Fecal R37.11.1 Farm Cow Dry Cow 99.7% 99.8% 99.8% 99.1% 99.7% RH Fecal R37.11.3 Farm Cow Dry Cow 100.0% 39.6% 99.6% 99.8% 100.0% RH Fecal R37.11.4 Farm Cow Dry Cow 99.9% 99.6% 99.5% 99.6% 5.9% RH Fecal R37.11.9 Farm Cow Dry Cow 51.2% 12.3% 1.1% 99.4% 100.0% RH Fecal R37.12.9 Farm Cow Dry Cow 27.6% 25.5% 6.5% 66.0% 14.7% RH Fecal S82.9.1 Farm TMR Low 22.3% 0.0% 0.0% 39.0% 10.2% RH R40.3.3 Farm Cow Bedpack 95.5% 7.6% 91.5% 94.9% 28.6% RG R40.4.4 Farm Cow Free Stall 99.8% 73.1% 4.0% 98.9% 99.5% RG R40.5.6 Farm Cow Free Stall 99.1% 98.2% 34.8% 99.1% 22.0% RG R40.5.7 Farm Cow Free Stall 78.3% 0.0% 0.0% 75.7% 73.2% RG R40.6.1 Farm Cow Free Stall 0.0% 0.0% 0.0% 0.0% 0.0% RG R40.6.8 Farm Cow Free Stall 99.6% 99.3% 97.4% 96.8% 98.4% RG R40.6.9 Farm Cow Free Stall 25.5% 0.0% 0.0% 3.2% 23.4% RG R41.4.3 Farm Cow Mature 99.6% 98.9% 98.2% 96.8% 95.0% RR R41.4.6 Farm Cow Mature 27.1% 13.1% 27.1% 21.6% 18.0% RR R41.7.8 Farm Cow Mature 0.0% 0.0% 48.6% 77.2% 41.6% RR R41.8.1 Farm Cow Mature 0.0% 3.1% 0.0% 2.9% 0.0% RR R41.8.5 Farm Cow Mature 70.3% 55.2% 45.4% 38.8% 61.7% RR R41.8.6 Farm Cow Mature 59.9% 35.4% 28.8% 84.4% 51.0% RR R41.9.5 Farm Cow Mature 22.7% 0.0% 0.0% 0.0% 1.5% RR R41.9.9 Farm Cow Mature 78.6% 29.6% 1.8% 85.8% 66.9% RR R41.9.10 Farm Cow Mature 32.5% 43.5% 19.6% 0.0% 70.9% RR R41.10.2 Farm Cow Mature 88.2% 6.4% 0.0% 71.9% 28.5% RR R41.10.4 Farm Cow Mature 0.0% 0.0% 0.0% 0.0% 0.0% RR R41.10.10 Farm Cow Mature 29.0% 18.2% 8.9% 40.0% 43.6% RR R41.11.1 Farm Cow Mature 99.8% 16.2% 2.7% 14.0% 36.5% RR R41.11.5 Farm Cow Mature 99.8% 9.4% 0.0% 100.0% 99.8% RR R42.1.4 Farm B Calf Calf 100.0% 98.3% 100.0% 100.0% 99.8% R42.1.5 Farm B Calf Calf 95.2% 74.5% 97.2% 99.3% 99.3% R42.1.9 Farm B Calf Calf 100.1% 21.7% 0.0% 96.6% 10.8% R42.3.8 Farm B Cows Heifer 97.6% 6.8% 97.8% 0.0% 97.2% R42.6.2 Farm B Cows Milk Cows 0.1% 0.0% 0.0% 1.3% 8.5% R42.7.1 Farm B Cows Milk Cows 100.0% 71.7% 79.7% 93.7% 97.6% R42.7.3 Farm B Cows Milk Cows 99.3% 98.8% 99.3% 99.3% 99.3% R42.7.8 Farm B Cows Milk Cows 89.0% 73.5% 72.6% 85.2% 82.1% R42.8.5 Farm B Cows Milk Cows 98.1% 11.6% 19.6% 98.5% 16.0% R42.8.8 Farm B Cows Milk Cows 100.0% 100.0% 100.0% 100.0% 99.9% R42.9.3 Farm B Cows Milk Cows 97.2% 58.6% 31.3% 96.9% 68.5% R42.11.7 Farm B Cows Dry Cows 100.0% 92.7% 100.0% 99.9% 50.0% R42.12.1 Farm B Cows Dry Cows 49.9% 0.0% 3.7% 41.0% 38.0% R43.1.5 Farm Cows Fresh 58.7% 6.8% 7.3% 80.7% 39.6% WH R43.2.5 Farm Cows Fresh 85.7% 0.0% 2.9% 89.9% 10.0% WH R43.3.1 Farm Cows Fresh 100.0% 4.9% 8.6% 100.0% 99.9% WH R43.3.2 Farm Cows Fresh 100.0% 0.0% 0.0% 100.0% 100.0% WH R43.3.4 Farm Cows Fresh 49.3% 29.4% 4.5% 99.2% 56.8% WH R43.6.10 Farm Cows Fresh 0.0% 0.0% 0.0% 0.0% 33.2% WH R43.8.3 Farm Cows Fresh 99.1% 99.7% 99.8% 97.2% 30.6% WH R43.9.7 Farm Cows Fresh 99.6% 99.6% 13.8% 99.6% 99.6% WH R43.10.1 Farm Cows Fresh 98.9% 36.9% 99.2% 99.3% 49.8% WH R43.11.4 Farm Cows Mid Lactation 98.8% 99.5% 99.3% 99.0% 99.3% WH R43.12.3 Farm Cows Mid Lactation 99.6% 99.5% 99.4% 99.7% 99.4% WH R43.13.10 Farm Cows Mid Lactation 53.4% 0.0% 99.3% 97.9% 100.0% WH R43.14.1 Farm Cows Mid Lactation 95.9% 95.4% 94.6% 94.4% 95.6% WH R43.14.4 Farm Cows Mid Lactation 99.4% 99.4% 98.9% 99.5% 99.5% WH R43.14.9 Farm Cows Mid Lactation 99.8% 99.4% 99.7% 99.8% 99.7% WH R43.15.4 Farm Cows Mid Lactation 99.5% 99.5% 99.6% 99.6% 99.4% WH R43.15.10 Farm Cows Mid Lactation 34.4% 41.3% 25.1% 87.9% 29.2% WH R43.16.5 Farm Cows Mid Lactation 99.2% 96.6% 0.0% 99.4% 99.6% WH R43.17.5 Farm Cows Mid Lactation 99.7% 99.1% 99.5% 99.7% 99.3% WH R43.18.1 Farm Cows High 99.5% 99.5% 99.7% 99.5% 99.6% WH Lactation R43.22.2 Farm Cows High 99.7% 99.5% 99.5% 99.4% 99.5% WH Lactation R43.22.6 Farm Cows High 99.4% 99.6% 99.4% 99.4% 99.6% WH Lactation R43.24.3 Farm Cows High 99.4% 99.4% 99.4% 97.2% 99.3% WH Lactation R43.27.1 Farm Cows High 99.7% 99.4% 83.8% 99.5% 99.7% WH Lactation R43.27.3 Farm Cows High 99.5% 99.5% 99.5% 99.7% 99.4% WH Lactation R43.27.10 Farm Cows High 99.2% 99.6% 99.5% 99.5% 99.4% WH Lactation R43.28.7 Farm Cows 2 yr. olds 99.7% 26.2% 97.6% 97.7% 99.6% WH R43.29.5 Farm Cows 2 yr. olds 99.6% 99.3% 97.3% 96.5% 99.5% WH R43.30.10 Farm Cows 2 yr. olds 99.4% 99.1% 97.3% 96.5% 98.8% WH R43.31.9 Farm Cows 2 yr. olds 99.1% 98.3% 97.1% 79.6% 98.0% WH R43.31.10 Farm Cows 2 yr. olds 99.4% 98.8% 98.0% 96.6% 98.4% WH R43.34.2 Farm Cows 2 yr. olds 99.4% 97.3% 98.6% 96.9% 97.9% WH R43.34.4 Farm Cows 2 yr. olds 99.4% 98.9% 99.1% 97.3% 98.1% WH R43.34.9 Farm Cows 2 yr. olds 99.4% 99.2% 98.3% 98.5% 99.3% WH R43.35.4 Farm Cows Late Lactation 99.5% 99.3% 98.6% 98.5% 99.7% WH R43.36.6 Farm Cows Late Lactation 99.0% 99.1% 98.0% 97.9% 99.3% WH R43.36.8 Farm Cows Late Lactation 99.0% 98.9% 98.3% 98.1% 99.5% WH R43.37.7 Farm Cows Late Lactation 99.2% 99.0% 98.7% 98.4% 99.5% WH R43.37.8 Farm Cows Late Lactation 99.2% 98.9% 98.9% 98.5% 99.5% WH R43.39.1 Farm Cows Late Lactation 99.6% 98.8% 99.5% 99.2% 99.6% WH R43.39.8 Farm Cows Late Lactation 99.3% 99.2% 99.5% 99.2% 99.6% WH R43.39.10 Farm Cows Late Lactation 99.8% 99.3% 99.2% 99.5% 99.3% WH R43.40.2 Farm Cows Late Lactation 99.6% 99.3% 99.3% 99.0% 99.8% WH R43.40.3 Farm Cows Late Lactation 99.6% 99.3% 99.0% 98.9% 99.4% WH R43.42.1 Farm Cows Late Dry 99.2% 99.2% 98.9% 98.8% 99.5% WH R43.42.9 Farm Cows Late Dry 99.6% 99.6% 99.4% 99.0% 99.6% WH R43.42.10 Farm Cows Late Dry 99.8% 99.7% 99.8% 99.4% 99.9% WH R43.43.7 Farm Cows Late Dry 99.6% 99.6% 99.8% 99.3% 99.6% WH R43.51.7 Farm Cows Late Dry 99.3% 99.6% 99.6% 99.2% 99.5% WH R43.55.6 Farm Cows Late Dry 99.7% 99.3% 99.7% 99.4% 99.6% WH R43.55.7 Farm Cows Late Dry 92.3% 95.2% 99.6% 98.2% 99.4% WH S86.2.1 Farm Alfalfa 90.5% 86.9% 87.9% 95.5% 83.4% RR Haylage S88.5.5 Farm TMR Fresh 99.8% 74.6% 98.3% 99.4% 98.9% WH S88.9.5 Farm TMR Late Lactation 99.8% 99.8% 99.9% 99.9% 99.9% WH R44.2.10 Farm Calf Calf 26.7% 0.0% 23.6% 10.2% 29.9% MD Fecal R44.3.5 Farm Calf Calf 82.7% 0.0% 29.3% 5.4% 0.0% MD Fecal R44.4.3 Farm Calf Calf 5.5% 0.0% 0.0% 0.0% 0.0% MD Fecal R44.9.6 Farm Cow Fresh 31.9% 21.4% 17.1% 12.1% 24.1% MD Fecal R44.9.9 Farm Cow Fresh 66.6% 14.9% 10.5% 3.7% 11.3% MD Fecal R44.11.3 Farm Cow Fresh 99.5% 27.3% 19.2% 99.5% 99.5% MD Fecal R44.12.1 Farm Cow Fresh 96.9% 98.5% 0.0% 35.0% 0.0% MD Fecal R44.15.3 Farm Cow Fresh 99.3% 99.3% 99.6% 99.4% 99.3% MD Fecal R44.16.7 Farm Cow Fresh 98.5% 51.9% 97.1% 98.8% 79.1% MD Fecal R44.16.10 Farm Cow Fresh 99.8% 98.9% 100.0% 98.9% 99.4% MD Fecal R44.17.6 Farm Cow Fresh 99.5% 99.6% 99.5% 99.6% 98.8% MD Fecal R44.17.8 Farm Cow Fresh 99.5% 98.7% 99.5% 99.5% 98.7% MD Fecal R44.22.7 Farm Cow Early 99.7% 99.7% 99.2% 99.7% 99.1% MD Fecal Lactation R44.23.9 Farm Cow Early 97.7% 31.9% 66.0% 85.3% 66.0% MD Fecal Lactation R44.27.5 Farm Cow Early 38.9% 41.8% 21.2% 67.5% 50.6% MD Fecal Lactation R44.28.2 Farm Cow Mid Lactation 0.0% 4.0% 0.0% 0.0% 0.0% MD Fecal R44.31.4 Farm Cow Mid Lactation 99.7% 90.5% 33.9% 99.3% 98.2% MD Fecal R44.31.5 Farm Cow Mid Lactation 35.6% 42.0% 22.6% 82.7% 58.0% MD Fecal R44.33.4 Farm Cow Mid Lactation 39.6% 0.0% 0.0% 0.0% 18.8% MD Fecal R44.37.9 Farm Cow Mid Lactation 29.5% 0.0% 19.2% 21.2% 17.5% MD Fecal R44.37.10 Farm Cow Mid Lactation 2.3% 0.0% 0.0% 0.0% 0.0% MD Fecal R44.43.7 Farm Cow Late 22.5% 0.0% 0.9% 0.0% 19.7% MD Fecal Lactation R44.45.7 Farm Cow Late Lactation 27.5% 4.6% 10.3% 8.4% 15.5% MD Fecal R44.46.7 Farm Cow Late Lactation 22.1% 0.0% 0.0% 20.8% 8.1% MD Fecal R44.72.4 Farm Cow Robot 21.0% 0.0% 7.0% 15.4% 37.9% MD Fecal S89.3.1 Farm Ryegrass 26.9% 4.3% 0.0% 13.1% 5.0% MD Haylage S89.15.3 Farm WOM 2.8% 0.0% 0.0% 8.3% 30.0% MD #1046 S89.20.7 Farm WOM 11.3% 0.0% 0.0% 21.0% 24.7% MD #1550 S89.20.9 Farm WOM 0.0% 0.0% 0.0% 0.0% 28.8% MD #1550

Example 11: Selection of Bacillus Strains to Inhibit Clostridium Perfringens and Non-Toxigenic Clostridia Isolated from Ruminant Fecal Samples. (Interstate-29 Corridor)

Introduction:

Clostridium is a genus of Gram-positive, spore-forming bacteria that are common residents of the gastrointestinal tract. A number of Clostridium species have been linked to enteric disease in ruminants including hemorrhagic bowel syndrome (HBS), a disease often correlated to elevated levels of Clostridium perfringens Type A. While most of the enteric diseases caused by clostridia are acute and occur sporadically in herds, in general, the prognosis is poor and the first sign of illness may be death. Based on recent results sub-acute enteric clostridia disease challenges may be a more wide spread issue than acute challenges. Due to a low success rate from treatment in acute disease challenges a more common, emphasis needs to be placed on prophylactic measures.

The purpose of this research was to characterize the distribution and diversity of clostridia in ruminants and ensure inhibition of these isolates using novel Bacillus strains as a method to control the clostridia populations.

Materials and Methods:

Fecal samples (411) from cows, heifers and calves gathered from 8 farms in the 1-29 Corridor region were diluted 1:10 with sterile peptone, heat shocked for 30 minutes at 60° C., enumerated in sterile peptone and pour plated on Tryptose Sulphite Cycloserine (TSC) agar with D-cycloserine (400 mg/L) to select for clostridia species. Agar plates were incubated at 37° C. anaerobically for 24 hours. If present, isolated sulphite-reducing colonies were counted for a total clostridia count (CFU/g) and representative isolates were picked into Reinforced clostridia Medium (RCM) (Oxoid, CM0149) and incubated anaerobically for 24 hours at 37° C. After 24 hours of incubation the cultures were transferred (10%) to Brain Heart Infusion (BHI) broth (BD, 211059) and incubated anaerobically for 24 hours at 37° C.

DNA extractions were performed in 96-well blocks containing 500 μl presumptive clostridia culture per well. Cells were harvested by centrifugation at 4,700 rpm for 10 minutes, the supernatant was removed. Cells were re-suspended in 500 μl of 50 mM of EDTA-2Na (pH=8.0). Aliquots of 300 μl of the suspended cells were transferred to a new 96-well block and combined with 20 μl of lysozyme from chicken egg white (Sigma, L6876) solution (100 mg/ml in 50 mM EDTA) to lyse bacterial cells. The 96-well block was incubated for 1 hour at 37° C. to lyse bacterial cells. Following the incubation 220 μl of lysis buffer (6 M Guanidine, 20% Triton-X 100, 10 mM Tris-HCL, pH 7.5) was added, mixed then incubated at room temperature for 15 minutes. Following the incubation 20 μl of Proteinase K (NEB, 800 U/mL) was added to each well, mixed and incubated at 55° C. for 30 minutes to degrade proteins. The cell lysate was then transferred to 96-well binding plate (Promega, A2278) and centrifuged at 4,700 rpms for 5 minutes. Flow through was discarded, three washes of the binding plate columns were executed centrifuging 750 μl of Column Wash Solution (Promega, A1318) at 4700 rpms for 1 minute and 30 seconds discarding flow through at the end of each spin. The binding plate was centrifuged for an additional 10 minutes at 4,700 rpm to remove any residual ethanol. A clean elution plate was then placed under the binding plate and DNA was eluted with 200 μl, pre-warmed (55° C.), Nuclease Free Water (Promega, P1195).

DNA was screened for toxin genes (α, β, ε, and ι) specific to C. perfringens using polymerase chain reaction (PCR). Amplification of toxin genes was executed using a multiplex PCR containing four primer sets (Yoo et al., 1997) (Table 1.) The PCR mixture contained 2.5 μl 10×PCR Buffer, 2 μl of 50 mM MgCl₂, 0.5 μM of each primer (Table 1.), 0.1 μl of Invitrogen™ Platinum™ Taq DNA Polymerase, 2.5 μl of DNA, sterile water was added to achieve 25 μl for a total reaction volume. The mixture underwent 5 minutes at 94° C., followed by 30 cycles of 94° C. for 1 minute, 55° C. for 1 minute, 72° C. for 1 minute finishing with a final elongation of 3 minutes at 72° C. PCR products were observed using a Fragment Analyzer (Advanced Analytics) to determine if amplification was achieved. If one or multiple toxin genes were observed a toxin type identification was assigned to each isolate based on their toxin gene profile (Songer, 1996). C. perfringens positive to total clostridia isolate ratio was used to calculate an estimated C. perfringens count based on the total clostridia count.

Unique strain-specific genetic fingerprints were generated using Random Amplification of Polymorphic DNA (RAPD) analysis on select isolates to determine diversity among fecal C. perfringens isolates. The PCR contained 5 μl of DNA, 2.5 μl RAPD primer 2 (10 μM) (Table 1.), and 17.5 μl of sterile water which was added to a Ready-To-Go RAPD Analysis Bead (Life Sciences, 27-9500-01). The mixture underwent 5 minutes at 95° C., followed by 45 cycles of 95° C. for 1 minute, 36° C. for 1 minute, 72° C. for 2 minutes finishing with a final elongation of 5 minutes at 72° C. PCR products observed on a Fragment Analyzer (Advanced Analytics) to determine amplification patterns and were imported into BioNumerics, bioinformatics software, for analysis. RAPD patterns were compared with a band based Dice correlation analysis method to determine the similarity between RAPD patterns as a way to monitor diversity between isolates.

Antimicrobial screening was done on C. perfringens isolates obtained from ruminant samples to gauge the effectiveness of the antimicrobial bacteriocin produced by the inventors' identified Bacillus strains 747, 1104, 1541, 1781, and 2018. Bacteriocin was harvested by growing each strain at 32° C. in a shaking incubator at 150 rpms for 24 hours in Brain Heart Infusion (BHI) broth. A 1% transfer of the 24-hour culture to fresh BHI broth was executed after incubation. The Bacillus were then incubated for 36-48 hours in a 32° C. shaking incubator at 150 rpms. The culture was then centrifuged at 14,000×g for 20 minutes, supernatant was then filtered with a 0.2 m filter to remove any residual cells.

A bacteriocin turbidity assay was executed by growing C. perfringens strains isolated from ruminant fecal samples in RCM for 24 hours, anaerobically, at 37° C. Overnight culture was transferred (1%) to sterile RCM and immediately used in the assay. For each C. perfringens isolate at least six wells were run in a sterile 48 well reaction plate, 600 μl inoculated culture (positive control), 600 μl inoculated RCM+70 μl bacteriocin (747, 1104, 1541, 1781, and 2018) and 670 RCM (un-inoculated, negative control). Plates were incubated anaerobically at 37° C. for 24 hours then read using a BioTek Epoch Microplate Spectrophotometer, readings were taken at a wavelength of 600 nm. Optical density readings from the negative controls were subtracted from all OD readings and percent inhibition was calculated using the positive control and each bacteriocin treatment.

To identify clostridia that did not have at least one toxin gene specific to C. perfringens, a PCR reaction was performed on the isolate DNA to amplify the 16S region of rDNA using primers 27F-YM and 1492R-Y (Table 1). This was done on 20% of the isolates that did not contain a toxin gene specific to C. perfringens. The PCR mixture contained 5 μl of 10×PCR Buffer, 2 μl of 50 mM MgCl₂, 1 μl of 50 mM dNTPs, 0.4 μM of each primer (Table 1.), 0.2 μl of Invitrogen™ Platinum™ Taq DNA Polymerase, 5 μl of DNA, and sterile water was added to achieve 50 μl for a total reaction volume. The mixture underwent 4 minutes at 95° C., followed by 35 cycles of 95° C. for 30 seconds, 50° C. for 30 seconds, 72° C. for 2 minutes finishing with a final elongation of 7 minutes at 72° C. A quality check was done on the amplification and PCR product was sent to gene wiz (https://www.genewiz.com) to obtain the sequences for the 16S genes. Sequences were compared to known typed bacterial strains obtained from EZbiocloud online electronic database (http://www.ezbiocloud.net/). Based on comparisons of these sequences a bacterial identification was assigned to the isolates.

Results:

Fecal samples, 411, were collect from eight 1-29 Corridor Regional farms (Minnesota, South Dakota, and Iowa) from which 3,471 presumptive clostridia isolates were isolated as representatives of the clostridial diversity in the 1-29 Corridor region (Table 21.).

Clostridia enumeration results indicated the average level of clostridia CFU/g across all calf fecal samples was 270,000 CFU/g with individual fecal samples ranging from <10 to 7,020,000 CFU/g. While the average level of clostridia CFU/g across all cow fecal samples was 38,100 CFU/g with individual fecal samples ranging from 5 to 6,970,000 CFU/g (FIG. 76.). Samples appeared to range in clostridia levels for both calves (FIG. 77.) and cows (FIG. 78.).

C. perfringens enumeration results displayed the average level of C. perfringens CFU/g across all calf fecal samples was 54,800 CFU/g with individual samples ranging from <10 to 764,000 CFU/g. While the average level of C. perfringens CFU/g across all cow fecal samples was 35,400 CFU/g with individual samples ranging from <10 to 6,970,000 CFU/g (FIG. 79.). Samples appeared to range in clostridia levels for both calves (FIG. 80.) and cows (FIG. 81.).

Analysis of the toxin multiplex PCR results displayed which isolates contained toxin genes specific to C. perfringens. A total of 3,471 presumptive clostridia isolates from fecal samples have been tested for the indicated C. perfringens toxin genes. Of the 3,471 clostridia isolates screened, 1,549 isolates (44.2%) tested positive for at least 1 of the toxin genes. From the 1,549 toxin-gene positive isolates 1,534 (99%) were identified as Type A (αtoxin only), however β, ε and ι, toxins were also detected in the clostridia isolates.

Gentic RAPD fingerprint patterns displayed diversity among the 1,547 isolates that successful amplified. The isolates tested were harvested from calf fecal, cow fecal and feed and did not cluster strictly based on the sample type or farm. Isolates formed 72 clusters based on 75% similarity according to the Dice correlation method. The largest cluster was 776 isolates which was 50.2% of the total dendrogram.

Representatives from the RAPD dendrogram were selected to capture the diversity of the C. perfringens population from this region and subjected to inhibition assays. Antimicrobial testing using the bacteriocin turbidity assay displayed good inhibition of most ruminant fecal C. perfringens isolates using bacteriocin harvested from 747, 1104, 1541, 1781, and 2018. The bacteriocin from at least one of the strains 747, 1104, 1541, 1781, and 2018 were able to inhibit the growth >60% of 138 of the 156 isolates tested representing a total of 92.0% inhibition of the C. perfringens population based on the dendrogram (Table 22.).

Out of the 3,443 isolates collected 1,922 isolates (55.8%) were found to be non-toxigenic clostridia. Sequencing representatives (n=399) from the non-toxigenic clostridia displayed two dominate clostridia groups the Clostridium bifermentans group (Paraclostridium bifermentans and P. benzoelyticum) and the Clostridium beijerinckii group (Clostridium diolis, Clostridium beijerinckii, Clostridium chromiireducens, Clostridium saccharoperbutylacetonicum, Clostridium puniceum, and Clostridium saccharobutylicum), the two main groups of the non-toxigenic clostridia group made up 61.7% of the non-toxigenic isolates (FIG. 82.).

Discussion:

Fecal samples were used as the most readily available sample type to estimate the level and obtain isolates of clostridia and C. perfringens within the digestive system of ruminants. From the 411 fecal samples collected throughout the 1-29 Corridor region all samples had detectable levels of clostridia. Many isolates harvested (1,549 isolates) from the ruminant samples contained a toxin gene specific to C. perfringens. C. perfringens was detected in 76% of the cow fecal samples and in 50% of the calf fecal samples. The high presence of clostridia and C. perfringens indicates the risk for sub-acute enteric clostridia disease challenges in most ruminants throughout the 1-29 Corridor. C. perfringens isolates were diverse according to the RAPD genetic fingerprints but were not specific to sample type or farm. Diverse representatives of C. perfringens were mostly inhibited (>60%) by at least one bacteriocin from the following strains 747, 1104, 1541, 1781 or 2018. From the 156 isolates tested 138 isolates were inhibited by greater than 60% by at least one of the strains, representing inhibition of 92.0% of the C. perfringens population based on representation from clusters on the RAPD dendrogram. This indicates the Bacillus strains 747, 1104, 1541, 1781 and 2018 can inhibit a wide range of diversity of C. perfringens isolates. The Bacillus strains are not limited to specific clostridia strain(s) like a vaccine which may be missing large groups of the clostridia populations based on the genetic diversity observed in the RAPD dendrogram. DNA sequencing of the non-toxigenic clostridia revealed two major identification of Clostridium species. C. bifermentans group, which is known to produce 1,3-propanediol (Leja et al., 2014; Myszka et al., 2012) and C. beijerinckii group known to produce butanol and acetone (Hou et al., 2017). The production of the metabolic end products of these species could be having an impact in the rumen, reducing performance parameters such as milk production within a dairy cow.

The Bacillus strains offer a prophylactic effect on the clostridia populations which may not only increase rumen efficiency leading to increased milk production, but prevent acute levels of C. perfringens reducing the occurrence of digestive deaths. The high prevalence of clostridia and C. perfringens in fecal samples collected suggests efficiency improvement opportunities in many ruminants throughout the 1-29 Corridor. This example displays the diversity of clostridia isolates from the ruminant fecal and feed samples collected from the 1-29 Corridor. The Bacillus strains tested 747, 1104, 1541, 1781 and 2018, could inhibit most of the clostridia diversity observed in the 1-29 Corridor. The product, in accordance with this embodiment of the present invention could inhibit toxigenic clostridia isolated from ruminants in the 1-29 Corridor indicating a benefit in rumen efficiency if fed to dairy cows as a direct fed microbial (DFM).

TABLE 21 Fecal samples, 411, were collected from I-29 Corridor regional dairies separated by farm (8) and age (cow or calf) which were enumerated for clostridia, tested for C. perfringens, isolates were genotyped and tested for inhibition. Farm Name State Cow Fecal Calf Fecal Farm B Minnesota 36 7 Farm P Minnesota 48 5 Farm K Minnesota 13 4 Farm R Iowa 82 5 Farm KC South Dakota 60 0 Farm N Minnesota 71 10 Farm S Minnesota 46 0 Farm C Minnesota 22 2 Total 378 33

TABLE 22 Bacteriocin assay results displaying each isolate tested and the source of that isolate, isolated from I-29 Corridor regional fecal or feed samples. Inhibition was calculated based on the percent of growth for each treatment well compared to the positive control. Sample Bacillus Strains Isolate ID Farm Type Group 747 1104 1541 1781 2018 R50.34.2 Farm B Cow Fecal 2 year Cow 92.6% 41.7% 92.3% 17.5% 92.0% R50.30.1 Farm B Cow Fecal 2 year Cow 86.2% 61.5% 68.4% 74.4% 69.1% R50.26.4 Farm B Cow Fecal Fresh Cow 87.2% 87.6% 87.2% 87.2% 87.0% R50.25.6 Farm B Cow Fecal Fresh Cow 86.1% 86.1% 86.4% 86.8% 86.6% R50.24.2 Farm B Cow Fecal Fresh Cow 91.6% 24.7% 91.1% 15.4% 90.9% R50.22.3 Farm B Cow Fecal Fresh Cow 91.8% 14.8% 89.6% 0.0% 91.2% R50.38.1 Farm B Cow Fecal Mature Cow 80.2% 80.5% 19.8% 80.7% 80.5% R50.7.1 Farm B Calf Fecal Calf 86.6% 87.5% 64.6% 87.0% 87.3% S97.16.2 Farm B Feed Grass Hay 74.6% 74.1% 73.7% 74.6% 74.6% R51.8.6 Farm P Cow Fecal Heifer 87.6% 74.3% 87.4% 88.0% 87.6% R51.25.2 Farm P Cow Fecal Fresh 90.1% 26.3% 90.8% 89.2% 90.1% R51.20.8 Farm P Cow Fecal Close-up 90.7% 0.0% 90.7% 0.0% 90.4% R51.32.9 Farm P Cow Fecal High 19.9% 1.4% 89.0% 0.0% 89.7% R51.32.6 Farm P Cow Fecal High 92.6% 92.4% 92.2% 92.7% 92.4% R51.31.2 Farm P Cow Fecal High 80.1% 80.8% 80.8% 80.5% 80.5% R51.28.2 Farm P Cow Fecal Fresh 86.2% 85.3% 85.6% 85.8% 85.3% R51.45.1 Farm P Cow Fecal Late 90.8% 17.6% 91.6% 91.2% 91.8% R51.44.2 Farm P Cow Fecal Late 92.5% 35.3% 92.3% 92.2% 91.9% R51.42.3 Farm P Cow Fecal Late 87.1% 41.0% 45.3% 85.2% 58.8% R51.40.7 Farm P Cow Fecal Late 23.6% 13.3% 88.3% 0.0% 88.8% R51.38.7 Farm P Cow Fecal High 91.6% 91.7% 91.3% 91.6% 91.4% R51.37.3 Farm P Cow Fecal High 91.8% 91.5% 90.8% 91.5% 91.5% R51.51.8 Farm P Cow Fecal Dry 88.4% 87.8% 86.8% 87.4% 88.4% R52.4.8 Farm K Calf Fecal Calf 26.0% 20.6% 9.9% 38.9% 18.3% R52.4.1 Farm K Calf Fecal Calf 38.6% 26.5% 0.0% 43.9% 30.3% R52.17.10 Farm K Cow Fecal Lactating 88.9% 91.8% 91.1% 91.7% 91.9% R52.17.7 Farm K Cow Fecal Lactating 87.5% 87.7% 80.1% 76.2% 84.0% R52.17.5 Farm K Cow Fecal Lactating 0.0% 0.0% 0.0% 0.0% 0.0% R52.17.2 Farm K Cow Fecal Lactating 91.0% 90.7% 90.9% 91.0% 91.0% R52.17.1 Farm K Cow Fecal Lactating 92.4% 92.3% 92.2% 92.3% 92.4% R52.15.9 Farm K Cow Fecal Lactating 62.5% 58.6% 61.6% 56.5% 64.7% R52.15.2 Farm K Cow Fecal Lactating 88.0% 88.2% 80.4% 83.1% 83.7% R52.14.5 Farm K Cow Fecal Lactating 78.1% 27.9% 9.3% 84.0% 61.8% S99.6.5 Farm K Feed Close Up 0.0% 1.3% 16.7% 0.0% 0.0% TMR R53.13.8 Farm R Cow Fecal Close-up 89.7% 35.6% 15.6% 80.7% 71.3% Heifer R53.7.10 Farm R Cow Fecal Close-up 92.8% 91.9% 90.5% 91.3% 91.6% Heifer R53.5.8 Farm R Calf Fecal Calf 90.5% 1.0% 89.0% 88.4% 90.5% R53.22.9 Farm R Cow Fecal Fresh Heifer 90.0% 89.2% 87.8% 88.3% 89.7% R53.22.2 Farm R Cow Fecal Fresh Heifer 87.1% 88.8% 86.7% 86.4% 88.8% R53.20.10 Farm R Cow Fecal Fresh Heifer 90.1% 89.2% 87.6% 88.8% 88.8% R53.19.3 Farm R Cow Fecal Fresh Heifer 91.4% 91.0% 90.4% 89.5% 90.7% R53.19.2 Farm R Cow Fecal Fresh Heifer 91.5% 90.3% 90.7% 89.9% 91.4% R53.15.7 Farm R Cow Fecal Close-up 90.7% 89.8% 90.4% 90.0% 90.6% Heifer R53.34.4 Farm R Cow Fecal High Cows 92.5% 91.9% 92.1% 92.3% 92.1% R53.28.8 Farm R Cow Fecal Close-up 88.6% 89.7% 89.4% 88.3% 89.3% Cows R53.26.8 Farm R Cow Fecal Close-up 91.6% 91.6% 91.3% 91.3% 91.4% Cows R53.25.8 Farm R Cow Fecal Fresh Heifer 91.2% 90.8% 90.7% 91.0% 90.8% R53.25.3 Farm R Cow Fecal Fresh Heifer 89.6% 89.4% 89.1% 89.2% 88.9% R53.44.7 Farm R Cow Fecal Heifer 90.4% 89.7% 89.7% 90.1% 89.9% R53.39.2 Farm R Cow Fecal High Cows 92.5% 92.8% 92.8% 93.0% 92.6% R53.38.9 Farm R Cow Fecal High Cows 89.3% 90.7% 90.1% 89.4% 90.5% R53.35.2 Farm R Cow Fecal High Cows 91.2% 91.6% 91.2% 90.6% 92.2% R53.35.1 Farm R Cow Fecal High Cows 91.7% 91.7% 91.7% 92.0% 92.0% R53.51.8 Farm R Cow Fecal Heifer 88.3% 90.6% 89.6% 89.0% 90.3% R53.48.10 Farm R Cow Fecal Heifer 91.8% 91.5% 91.6% 91.8% 91.9% R53.48.1 Farm R Cow Fecal Heifer 90.7% 90.7% 90.4% 90.8% 90.7% R53.47.8 Farm R Cow Fecal Heifer 90.8% 91.1% 90.9% 91.1% 91.2% R53.63.1 Farm R Cow Fecal High Cows 3.0% 1.7% 38.7% 30.6% 45.1% R53.62.8 Farm R Cow Fecal High Cows 25.6% 17.6% 92.4% 38.1% 92.6% R53.61.2 Farm R Cow Fecal Fresh Cows 90.0% 0.0% 90.0% 20.7% 26.6% R53.56.4 Farm R Cow Fecal Fresh Cows 0.0% 0.0% 90.7% 8.9% 91.0% R53.55.7 Farm R Cow Fecal Fresh Cows 90.7% 91.2% 90.7% 91.2% 91.3% R53.55.1 Farm R Cow Fecal Fresh Cows 13.5% 0.0% 28.6% 21.2% 28.5% R53.71.2 Farm R Cow Fecal Late Cows 0.0% 0.0% 47.3% 35.5% 44.4% R53.70.8 Farm R Cow Fecal High Cows 0.0% 0.0% 0.0% 23.8% 36.3% R53.69.5 Farm R Cow Fecal High Cows 9.0% 0.0% 8.3% 41.0% 48.6% R53.66.3 Farm R Cow Fecal High Cows 92.1% 87.6% 92.2% 11.8% 92.4% R53.66.2 Farm R Cow Fecal High Cows 11.5% 0.0% 37.7% 19.5% 45.2% R53.75.4 Farm R Cow Fecal Late Cows 64.5% 0.0% 61.6% 0.0% 40.1% S102.7.3 Farm R Feed Fresh Heifer 80.3% 1.3% 63.3% 74.4% 76.7% TMR S102.1.10 Farm R Feed Baylage 58.5% 62.1% 66.4% 57.4% 57.3% Mark S102.1.9 Farm R Feed Baylage 49.2% 51.3% 43.4% 50.9% 53.4% Mark S102.1.3 Farm R Feed Baylage 35.8% 44.2% 45.8% 29.2% 41.2% Mark R53.88.10 Farm R Cow Fecal Late Cows 90.5% 80.5% 90.7% 90.7% 90.8% R53.88.6 Farm R Cow Fecal Late Cows 31.7% 22.7% 28.8% 17.1% 13.5% R53.87.3 Farm R Cow Fecal Late Cows 90.8% 88.0% 86.6% 91.6% 90.8% R53.85.8 Farm R Cow Fecal Late Cows 65.5% 70.4% 77.4% 66.3% 82.7% S102.11.1 Farm R Feed Fresh TMR 0.0% 11.7% 0.0% 0.0% 0.0% S102.8.8 Farm R Feed Close Up 80.5% 59.8% 80.0% 85.2% 85.4% Cow TMR S102.8.5 Farm R Feed Close Up 59.2% 40.0% 71.6% 62.4% 55.8% Cow TMR S102.8.4 Farm R Feed Close Up 67.8% 49.4% 85.5% 71.1% 65.3% Cow TMR R59.8.5 Farm Cow Fecal 2+ 95.8% 97.8% 94.2% 97.3% 97.3% KC R59.8.4 Farm Cow Fecal 2+ 99.3% 98.9% 99.5% 99.5% 99.6% KC R59.7.7 Farm Cow Fecal 2+ 98.9% 99.6% 98.9% 99.2% 98.9% KC R59.6.4 Farm Cow Fecal 2+ 99.6% 99.1% 99.6% 99.8% 99.6% KC R59.8.10 Farm Cow Fecal 2+ 88.7% 3.1% 4.2% 88.7% 62.1% KC R59.22.3 Farm Cow Fecal Early Lac. 99.9% 99.9% 99.7% 99.9% 99.7% KC R59.15.9 Farm Cow Fecal Mid/Late 95.0% 92.5% 75.0% 96.3% 73.8% KC Lac. R59.39.8 Farm Cow Fecal Fresh 99.1% 98.4% 99.4% 99.1% 99.1% KC R59.38.9 Farm Cow Fecal Fresh 99.6% 99.6% 99.7% 99.7% 99.9% KC R59.37.5 Farm Cow Fecal Fresh 97.1% 97.1% 97.1% 97.3% 97.3% KC R59.35.9 Farm Cow Fecal Fresh 99.3% 98.6% 98.9% 99.3% 98.9% KC R59.33.8 Farm Cow Fecal Fresh 97.4% 97.6% 97.4% 96.3% 97.6% KC R59.50.7 Farm Cow Fecal 1st Lac. 97.7% 99.7% 98.3% 97.0% 98.1% KC R59.48.10 Farm Cow Fecal 1st Lac. 97.7% 99.7% 99.6% 99.6% 99.4% KC R59.48.8 Farm Cow Fecal 1st Lac. 99.7% 99.7% 99.9% 98.7% 99.9% KC R59.47.6 Farm Cow Fecal 1st Lac. 94.2% 87.9% 77.8% 86.0% 93.7% KC R59.47.3 Farm Cow Fecal 1st Lac. 95.4% 95.2% 78.3% 95.8% 85.2% KC R59.47.2 Farm Cow Fecal 1st Lac. 99.5% 99.7% 99.3% 99.3% 99.7% KC R59.59.3 Farm Cow Fecal Close Up 99.7% 99.9% 99.7% 99.6% 99.7% KC R59.59.2 Farm Cow Fecal Close Up 97.8% 97.8% 97.9% 97.8% 97.9% KC R59.58.6 Farm Cow Fecal Close Up 96.6% 96.6% 96.6% 96.3% 96.3% KC R62.20.6 Farm S Cow Fecal Pregnant 66.7% 56.7% 56.7% 63.3% 60.0% R62.24.5 Farm S Cow Fecal Pregnant 96.0% 95.5% 95.5% 95.7% 96.0% R62.29.6 Farm S Cow Fecal Pregnant 97.6% 97.9% 97.9% 97.6% 97.6% R62.32.2 Farm S Cow Fecal Dry Cow 96.0% 95.6% 95.6% 96.5% 96.5% R62.5.1 Farm S Cow Fecal Breeding 2+ 100.0% 99.7% 99.9% 100.0% 99.7% R62.9.2 Farm S Cow Fecal Breeding 2+ 98.9% 98.2% 99.0% 99.0% 98.2% R63.19.9 Farm S Cow Fecal 1st 98.7% 98.7% 96.0% 98.5% 98.5% Lactation Breeding R63.21.3 Farm S Cow Fecal Pregnant 85.7% 70.1% 84.4% 87.0% 70.1% R63.22.3 Farm S Cow Fecal Pregnant 97.8% 97.0% 98.2% 95.7% 98.2% R63.23.3 Farm S Cow Fecal Pregnant 98.7% 98.7% 98.8% 98.7% 98.7% R63.23.9 Farm S Cow Fecal Pregnant 96.0% 97.0% 98.4% 96.1% 96.9% R63.27.5 Farm S Cow Fecal Pregnant 97.9% 98.3% 98.1% 95.0% 98.5% R63.28.1 Farm S Cow Fecal Pregnant 96.8% 97.1% 96.8% 97.1% 97.1% R63.1.7 Farm C Cow Fecal Lactation 98.7% 99.2% 99.1% 99.1% 99.4% Pen 2 R63.11.6 Farm C Cow Fecal Lactation 99.6% 99.4% 99.2% 99.4% 99.6% Pen 1 R63.18.9 Farm C Cow Fecal Lactation 99.1% 97.8% 96.8% 97.8% 98.0% Pen 1 R63.2.6 Farm C Cow Fecal Lactation 16.8% 1.5% 25.7% 48.9% 35.0% Pen 2 R63.32.1 Farm C Cow Fecal Close Up 99.8% 99.5% 99.7% 99.8% 99.7% R63.34.3 Farm C Calf Fecal Calves 12.0% 47.2% 23.7% 41.2% 48.9% R63.34.4 Farm C Calf Fecal Calves 95.1% 90.6% 88.5% 96.2% 95.5% R63.34.5 Farm C Calf Fecal Calves 97.4% 19.2% 11.7% 92.3% 87.8% R63.4.1 Farm C Cow Fecal Lactation 98.5% 68.6% 98.5% 99.0% 98.8% Pen 2 R63.6.4 Farm C Cow Fecal Lactation 99.4% 52.3% 99.2% 98.9% 99.4% Pen 2 R63.9.2 Farm C Cow Fecal Lactation 99.5% 99.5% 99.0% 99.5% 98.5% Pen 2 S111.4.9 Farm C Lactation Lactation 75.7% 73.4% 99.5% 80.1% 92.3% 1 TMR Pen 1 R60.12.10 Farm N Cow Fecal Late 89.2% 30.3% 66.2% 0.0% 0.0% R60.14.6 Farm N Cow Fecal Late 99.9% 99.8% 99.6% 99.6% 99.8% R60.15.9 Farm N Cow Fecal Late 99.6% 98.9% 99.2% 98.8% 99.4% R60.17.6 Farm N Cow Fecal Late 98.0% 93.5% 98.0% 92.8% 98.0% R60.2.2 Farm N Calf Fecal Calves 0.0% 0.0% 42.9% 0.0% 0.0% R60.21.4 Farm N Cow Fecal High 96.8% 33.9% 89.5% 55.2% 67.0% R60.22.8 Farm N Cow Fecal High 99.5% 99.1% 99.4% 99.1% 99.4% R60.30.3 Farm N Cow Fecal High 94.9% 94.8% 90.2% 94.8% 98.3% R60.31.3 Farm N Cow Fecal High 61.8% 69.4% 70.5% 66.3% 60.1% R60.38.2 Farm N Cow Fecal Heifer 99.9% 99.5% 99.7% 99.7% 99.5% R60.4.3 Farm N Calf Fecal Calves 99.6% 99.0% 99.2% 99.4% 99.2% R60.46.10 Farm N Cow Fecal Close Up 94.6% 94.6% 96.4% 82.1% 92.5% R60.52.1 Farm N Cow Fecal Fresh 98.3% 79.9% 72.7% 97.1% 95.2% R60.52.7 Farm N Cow Fecal Fresh 98.3% 99.3% 97.9% 99.4% 99.5% R60.59.7 Farm N Cow Fecal Fresh 89.5% 92.3% 92.3% 91.7% 91.7% R60.59.9 Farm N Cow Fecal Fresh 99.6% 99.7% 99.6% 99.7% 99.7% R60.61.2 Farm N Cow Fecal High 99.8% 99.8% 99.8% 99.6% 99.6% R60.64.1 Farm N Cow Fecal High 99.8% 99.4% 99.5% 99.6% 99.8% R60.64.2 Farm N Cow Fecal High 97.9% 97.9% 97.9% 96.7% 98.5% R60.64.3 Farm N Cow Fecal High 99.8% 99.6% 99.6% 99.8% 99.7% R60.64.8 Farm N Cow Fecal High 0.0% 0.0% 0.0% 0.0% 0.0% R60.66.1 Farm N Cow Fecal High 99.2% 99.0% 98.9% 98.1% 99.4% R60.70.4 Farm N Cow Fecal High 99.1% 98.7% 84.2% 99.1% 98.1% R60.70.8 Farm N Cow Fecal High 98.0% 51.3% 58.0% 90.0% 86.0% R60.71.10 Farm N Cow Fecal High 99.4% 99.4% 99.6% 99.7% 99.6% R60.72.1 Farm N Cow Fecal Close Up 99.5% 99.5% 99.5% 99.6% 99.2% R60.72.2 Farm N Cow Fecal Close Up 98.7% 98.4% 98.4% 98.5% 98.7% R60.73.1 Farm N Cow Fecal Close Up 94.7% 98.6% 99.0% 99.0% 99.0% R60.81.8 Farm N Cow Fecal Close Up 96.3% 97.8% 98.3% 98.5% 98.8%

Example 12: The Effect of a Combination of Bacillus Strains on the Clostridial Populations of Dairy Cows on Farm ALJ in Texas

Introduction: Hemorrhagic bowel syndrome (HBS) was first reported in 1991 and observed in five high-producing Holstein cows from one dairy in Idaho (Sockett, 2004). Symptoms included point-source sub-mucosal hematomas, each affecting 10-20 cm of the jejunum. One of the five cows exhibited a ruptured hematoma with exsanguination into the lumen of the jejunum. Although Aspergillus fumigatus and Clostridium perfringens are known to be involved in the etiology of HBS, the syndrome is better described as being poly-microbial and multi-factorial in nature. Increased consumption of a high-energy diet seems to be the most plausible common pathway for all the risk factors that have been described (Berghaus et al., 2005).

HBS is characterized by sudden drop in milk production, abdominal pain due to obstructed bowel and anemia (Anderson, 2002). Clinical signs of the disease are decreased feed intake, depression, decreased milk production, dehydration, abdominal distension and dark clotted blood in the feces. Death comes within 48 hours from the onset of the obstructing blood clot plug. Due to the sporadic, acute etiology few treatments are known to be effective

In addition to Clostridium isolates of several species causing enteric disease, other Clostridium species produce high levels of acetone, butanol, 1,3 propanediol and butyric acid as end products of their metabolism. If produced in the rumen, these metabolic end products may affect rumen function and decrease efficiency.

The Bacillus strains selected by the inventors, to inhibit pathogens, produce multiple compounds with inhibitory activity against other microbes with many strains containing more than ten operons producing antifungal and antibacterial compounds. Multiple bacteriocins are being produced in vitro directly at the site of action by the Bacillus strains so a robust blend of bacteriocins are present at doses lower than would be needed if isolated bacteriocins were being added directly to the feed.

The purpose of this study was to measure the levels and diversity of clostridia in dairy cows on Farm ALJ treated with the product, in accordance with this embodiment of the present invention, (referred to herein as “treated”), over 110 days.

Materials and Methods:

A dairy herd in Texas (Farm ALJ) was selected to study the impact of Bacillus product, in accordance with this embodiment of the present invention, on clostridial levels and diversity. The herd consists of 2900 milk cows housed in a Saudi style barns and bedded on sand.

The product, in accordance with this embodiment of the present invention was a combination product of two Bacillus strains in equal proportions; Bacillus 747 and Bacillus 1781 incorporated into the total mixed ration (TMR) at a dose of 2 billion CFU per head per day.

Fecal samples were obtained from 90 cows at two time periods before treatment and 63 cows after 110 days on treatment.

Fecal samples from cows were diluted 1:10 with sterile peptone, heat shocked for 30 minutes at 60° C., enumerated in sterile peptone and pour plated on Tryptose Sulphite Cycloserine (TSC) agar with D-cycloserine (400 mg/L) to select for clostridial species. Agar plates were incubated at 37° C. anaerobically for 24 hours. If present, isolated sulphite-reducing colonies were counted for total clostridia counts (CFU/g) and representative isolates were picked into Reinforced Clostridia Medium (RCM) (Oxoid, CM0149) and incubated anaerobically for 24 hours at 37° C. After 24 hours of incubation the cultures were transferred (10%) to Brain Heart Infusion (BHI) broth (BD, 211059) and incubated anaerobically for 24 hours at 37° C.

DNA extractions were performed in 96-well blocks containing 500 μl presumptive clostridia culture per well. Cells were harvested by centrifugation at 4,700 rpm for 10 minutes, the supernatant was removed. Cells were re-suspended in 500 μl of 50 mM of EDTA-2Na (pH=8.0). Aliquots of 300 μl of the suspended cells were transferred to a new 96-well block and combined with 20 μl of lysozyme from chicken egg white (Sigma, L6876) solution (100 mg/ml in 50 mM EDTA) to lyse bacterial cells. The 96-well block was incubated for 1 hour at 37° C. to lyse bacterial cells. Following the incubation 220 μl of lysis buffer (6 M Guanidine, 20% Triton-X 100, 10 mM Tris-HCL, pH 7.5) was added, mixed then incubated at room temperature for 15 minutes. Following the incubation 20 μl of Proteinase K (NEB, 800 U/mL) was added to each well, mixed and incubated at 55° C. for 30 minutes to degrade proteins. The cell lysate was then transferred to 96-well binding plate (Promega, A2278) and centrifuged at 4,700 rpms for 5 minutes. Flow through was discarded, three washes of the binding plate columns were executed centrifuging 750 μl of Column Wash Solution (Promega, A1318) at 4700 rpms for 1 minute and 30 seconds discarding flow through at the end of each spin. The binding plate was centrifuged for an additional 10 minutes at 4,700 rpm to remove any residual ethanol. A clean elution plate was then placed under the binding plate and DNA was eluted with 200 μl, pre-warmed (55° C.), Nuclease Free Water (Promega, P1195).

DNA was screened for toxin genes (α, β, ε, and ι) specific to C. perfringens using polymerase chain reaction (PCR). Amplification of toxin genes was executed using a multiplex PCR containing four primer sets (Yoo et al., 1997) (Table 1.) The PCR mixture contained 2.5 μl 10×PCR Buffer, 2 μl of 50 mM MgCl₂, 0.5 μM of each primer (Table 1.), 0.1 μl of Invitrogen™ Platinum™ Taq DNA Polymerase, 2.5 μl of DNA, sterile water was added to achieve 25 μl for a total reaction volume. The mixture underwent 5 minutes at 94° C., followed by 30 cycles of 94° C. for 1 minute, 55° C. for 1 minute, 72° C. for 1 minute finishing with a final elongation of 3 minutes at 72° C. PCR products were observed using a Fragment Analyzer (Advanced Analytics) to determine if amplification was achieved. If one or multiple toxin genes were observed a toxin type identification was assigned to each isolate based on their toxin-gene profile (Songer, 1996). C. perfringens positive to total clostridia isolate ratio was used to calculate an estimated C. perfringens count based on the total clostridia count.

Unique strain-specific genetic fingerprints were generated using Random Amplification of Polymorphic DNA (RAPD) analysis on select isolates to determine diversity among fecal C. perfringens isolates. The PCR contained 5 μl of DNA, 2.5 μl RAPD primer 2 (10 μM) (Table 1.), and 17.5 μl of sterile water which was added to a Ready-To-Go RAPD Analysis Bead (Life Sciences, 27-9500-01). The mixture underwent 5 minutes at 95° C., followed by 45 cycles of 95° C. for 1 minute, 36° C. for 1 minute, 72° C. for 2 minutes finishing with a final elongation of 5 minutes at 72° C. PCR products observed on a Fragment Analyzer (Advanced Analytics) to determine amplification patterns and were imported into BioNumerics, bioinformatics software, for analysis. RAPD patterns were compared with a band based Dice correlation analysis method to determine the similarity between RAPD patterns as a way to monitor diversity between isolates. Cluster cut-off was at 75% similarity.

To identify clostridia that did not have at least one toxin gene specific to C. perfringens, a PCR reaction was performed on the isolate DNA to amplify the 16S region of rDNA using primers 27F-YM and 1492R-Y (Table 1). This was done on 20% of the isolates that did not contain a toxin gene specific to C. perfringens. The PCR mixture contained 5 μl of 10×PCR Buffer, 2 μl of 50 mM MgCl₂, 1 μl of 50 mM dNTPs, 0.4 μM of each primer (Table 1.), 0.2 μl of Invitrogen™ Platinum™ Taq DNA Polymerase, 5 μl of DNA, and sterile water was added to achieve 50 μl for a total reaction volume. The mixture underwent 4 minutes at 95° C., followed by 35 cycles of 95° C. for 30 seconds, 50° C. for 30 seconds, 72° C. for 2 minutes finishing with a final elongation of 7 minutes at 72° C. A quality check was done on the amplification and PCR product was sent to Genewiz (https://www.genewiz.com) to obtain the sequences for the 16S genes. Sequences were compared to known type strains obtained from EZbiocloud online electronic database (http://www.ezbiocloud.net/). Based on comparisons of these sequences a bacterial identification was assigned to the isolates.

Results:

Total clostridia counts on Farm ALJ (FIG. 83) were not significantly different than those determined during the Texas survey (Example 6). During the 110 days on the product total clostridia decreased significantly. Although there was no reduction in C. perfringens treatment with the Bacillus blend (FIG. 84), there was less strain diversity in the C. perfringens isolated during treatment as indicated by the Shannon-Wiever index of the C. perfringens RAPD patterns (Table 23). Diversity of the non-toxigenic clostridia was also reduced during treatment and the predominant C. beijerincki group isolates were supplanted by C. tertium group isolates during treatment (FIG. 85 and Table 24).

Discussion:

The blend of Bacillus strains caused a decrease in total clostridial counts and decreased the diversity of C. perfringens strains in the cows. The Bacillus product also reduced the diversity of non-toxigenic clostridial species and caused the displacement of C. beijerinckii group strains by C. tertium group. These data demonstrate that the product causes a reduction in clostridial counts and a reduction in the diversity of C. perfringens isolates and the diversity and types of Clostridium species. The reduction in the proportion of the C. beijerinckii group, which are known to produce high levels of butanol and acetone, will most likely improve rumen fermentation and improve feed efficiency as well as milk production in dairy cows.

TABLE 23 RAPD fingerprint diversity of the Clostridium perfringens isolates over time. Shannon-Wiener Isolates Clusters Index of Diversity Pretreatment 279 44 2.88 Treatment Day 110 256 23 2.11

TABLE 24 Diversity of non-toxigenic Clostridium species and change in proportions of the two most predominant groups over time. Shannon- Wiener Clostridium Clostridium Species Iso- Spe- Index of beijerinckii tertium Diversity lates cies Diversity group group Pretreatment 36 11 2.09 53%  6% Day 110 30 5 1.26  0% 40%

Example 13: The Effect of a Combination of Bacillus Strains on the Clostridial Populations of Dairy Cows on Farm E in Wisconsin

Introduction:

Hemorrhagic bowel syndrome (HBS) was first reported in 1991 and observed in five high-producing Holstein cows from one dairy in Idaho (Sockett, 2004). Symptoms included point-source sub-mucosal hematomas, each affecting 10-20 cm of the jejunum. One of the five cows exhibited a ruptured hematoma with exsanguination into the lumen of the jejunum. Although Aspergillus fumigatus and Clostridium perfringens are known to be involved in the etiology of HBS, the syndrome is better described as being poly-microbial and multi-factorial in nature. Increased consumption of a high-energy diet seems to be the most plausible common pathway for all the risk factors that have been described (Berghaus et al., 2005).

HBS is characterized by sudden drop in milk production, abdominal pain due to obstructed bowel and anemia (Anderson, 2002). Clinical signs of the disease are decreased feed intake, depression, decreased milk production, dehydration, abdominal distension and dark clotted blood in the feces. Death comes within 48 hours from the onset of the obstructing blood clot plug. Due to the sporadic, acute etiology few treatments are known to be effective

In addition to Clostridium isolates of several species causing enteric disease, other Clostridium species produce high levels of acetone, butanol, 1,3 propanediol and butyric acid as end products of their metabolism. If produced in the rumen, these metabolic end products may affect rumen function and decrease efficiency.

The Bacillus strains selected by the inventors, to inhibit pathogens, produce multiple compounds with inhibitory activity against other microbes with many strains containing more than ten operons producing antifungal and antibacterial compounds. Multiple bacteriocins are being produced in vitro directly at the site of action by the Bacillus strains so a robust blend of bacteriocins are present at doses lower than would be needed if isolated bacteriocins were being added directly to the feed.

The purpose of this study was to measure the levels and diversity of clostridia in dairy cows on Wisconsin Farm E treated with the product, in accordance with this embodiment of the present invention, (referred to herein as “treated”), over 220 days.

Materials and Methods:

A dairy herd in Wisconsin (Farm E) was selected to study the impact of a Bacillus product, in accordance with this embodiment of the present invention, on clostridial level and diversity. The herd consists of 650 milk cows housed in a free stall barn and the cows are bedded on sand.

The product, in accordance with this embodiment of the present invention was a combination product of three Bacillus strains; Bacillus 747 (50%), Bacillus 1781 (45%) and Bacillus 1541 (5%) incorporated into the total mixed ration (TMR) at a dose of about 2 billion CFU per head per day.

Fecal samples were obtained from cows before and during treatment. Sample dates and number of animals sampled are indicated in Table 25.

Fecal samples from cows were diluted 1:10 with sterile peptone, heat shocked for 30 minutes at 60° C., enumerated in sterile peptone and pour plated on Tryptose Sulphite Cycloserine (TSC) agar with D-cycloserine (400 mg/L) to select for clostridial species. Agar plates were incubated at 37° C. anaerobically for 24 hours. If present, isolated sulphite-reducing colonies were counted for total clostridia counts (CFU/g) and representative isolates were picked into Reinforced Clostridia Medium (RCM) (Oxoid, CM0149) and incubated anaerobically for 24 hours at 37° C. After 24 hours of incubation the cultures were transferred (10%) to Brain Heart Infusion (BHI) broth (BD, 211059) and incubated anaerobically for 24 hours at 37° C.

DNA extractions were performed in 96-well blocks containing 500 μl presumptive clostridia culture per well. Cells were harvested by centrifugation at 4,700 rpm for 10 minutes, the supernatant was removed. Cells were re-suspended in 500 μl of 50 mM of EDTA-2Na (pH=8.0). Aliquots of 300 μl of the suspended cells were transferred to a new 96-well block and combined with 20 μl of lysozyme from chicken egg white (Sigma, L6876) solution (100 mg/ml in 50 mM EDTA) to lyse bacterial cells. The 96-well block was incubated for 1 hour at 37° C. to lyse bacterial cells. Following the incubation 220 μl of lysis buffer (6 M Guanidine, 20% Triton-X 100, 10 mM Tris-HCL, pH 7.5) was added, mixed then incubated at room temperature for 15 minutes. Following the incubation 20 μl of Proteinase K (NEB, 800 U/mL) was added to each well, mixed and incubated at 55° C. for 30 minutes to degrade proteins. The cell lysate was then transferred to 96-well binding plate (Promega, A2278) and centrifuged at 4,700 rpms for 5 minutes. Flow through was discarded, three washes of the binding plate columns were executed centrifuging 750 μl of Column Wash Solution (Promega, A1318) at 4700 rpms for 1 minute and 30 seconds discarding flow through at the end of each spin. The binding plate was centrifuged for an additional 10 minutes at 4,700 rpm to remove any residual ethanol. A clean elution plate was then placed under the binding plate and DNA was eluted with 200 μl, pre-warmed (55° C.), Nuclease Free Water (Promega, P1195).

DNA was screened for toxin genes (α, β, ε, and ι) specific to C. perfringens using polymerase chain reaction (PCR). Amplification of toxin genes was executed using a multiplex PCR containing four primer sets (Yoo et al., 1997) (Table 1.) The PCR mixture contained 2.5 μl 10×PCR Buffer, 2 μl of 50 mM MgCl₂, 0.5 μM of each primer (Table 1.), 0.1 μl of Invitrogen™ Platinum™ Taq DNA Polymerase, 2.5 μl of DNA, sterile water was added to achieve 25 μl for a total reaction volume. The mixture underwent 5 minutes at 94° C., followed by 30 cycles of 94° C. for 1 minute, 55° C. for 1 minute, 72° C. for 1 minute finishing with a final elongation of 3 minutes at 72° C. PCR products were observed using a Fragment Analyzer (Advanced Analytics) to determine if amplification was achieved. If one or multiple toxin genes were observed a toxin type identification was assigned to each isolate based on their toxin-gene profile (Songer, 1996). C. perfringens positive to total clostridia isolate ratio was used to calculate an estimated C. perfringens count based on the total clostridia count.

Unique strain-specific genetic fingerprints were generated using Random Amplification of Polymorphic DNA (RAPD) analysis on select isolates to determine diversity among fecal C. perfringens isolates. The PCR contained 5 μl of DNA, 2.5 μl RAPD primer 2 (10 μM) (Table 1.), and 17.5 μl of sterile water which was added to a Ready-To-Go RAPD Analysis Bead (Life Sciences, 27-9500-01). The mixture underwent 5 minutes at 95° C., followed by 45 cycles of 95° C. for 1 minute, 36° C. for 1 minute, 72° C. for 2 minutes finishing with a final elongation of 5 minutes at 72° C. PCR products observed on a Fragment Analyzer (Advanced Analytics) to determine amplification patterns and were imported into BioNumerics, bioinformatics software, for analysis. RAPD patterns were compared with a band based Dice correlation analysis method to determine the similarity between RAPD patterns as a way to monitor diversity between isolates. Cluster cut-off was at 75% similarity.

To identify clostridia that did not have at least one toxin gene specific to C. perfringens, a PCR reaction was performed on the isolate DNA to amplify the 16S region of rDNA using primers 27F-YM and 1492R-Y (Table 1). This was done on 20% of the isolates that did not contain a toxin gene specific to C. perfringens. The PCR mixture contained 5 μl of 10×PCR Buffer, 2 μl of 50 mM MgCl₂, 1 μl of 50 mM dNTPs, 0.4 μM of each primer (Table 1.), 0.2 μl of Invitrogen™ Platinum™ Taq DNA Polymerase, 5 μl of DNA, and sterile water was added to achieve 50 μl for a total reaction volume. The mixture underwent 4 minutes at 95° C., followed by 35 cycles of 95° C. for 30 seconds, 50° C. for 30 seconds, 72° C. for 2 minutes finishing with a final elongation of 7 minutes at 72° C. A quality check was done on the amplification and PCR product was sent to Genewiz (https://www.genewiz.com) to obtain the sequences for the 16S genes. Sequences were compared to known type strains obtained from EZbiocloud online electronic database (http://www.ezbiocloud.net/). Based on comparisons of these sequences a bacterial identification was assigned to the isolates.

Results:

Total clostridia counts on Farm E (FIG. 86) were not significantly different than those determined during the Wisconsin survey (Example 5). Although there was no reduction in clostridia (FIG. 86) or C. perfringens (FIG. 87) during treatment with the Bacillus blend, there was less strain diversity in the C. perfringens isolated during treatment as indicated by the Shannon-Wiever index of the C. perfringens RAPD patterns (Table 26). Diversity of the non-toxigenic clostridia was also reduced during treatment and the predominant C. beijerincki group isolates were totally supplanted by C. bifermentans group isolates during treatment (FIG. 88 and Table 27).

Discussion:

The blend of Bacillus strains selected to inhibit C. perfringens decreased the diversity of C. perfringens strains in the cows. The Bacillus product also reduced the diversity of non-toxigenic clostridial species and caused the displacement of C. beijerinckii group strains by C. bifermentans and others. These data demonstrate that even when clostridical counts are within the normal range for the region and are not reduced by the product there is still a reduction in the diversity of C. perfringens isolates and the diversity and types of Clostridium species present. C. beijerinckii group was predominant at 96% before treatment, but was not detected after 220 days on treatment. The reduction in the proportion of the C. beijerinckii group, which are known to produce high levels of butanol and acetone, will most likely improve rumen fermentation and improve feed efficiency as well as milk production in dairy cows.

TABLE 25 Number of fecal samples collected at each sampling point from Wisconsin Farm E. Samples Date Cow Fecal Pretreatment May 11, 2016 40 Treatment Day 60 Jul. 13, 2016 38 Treatment Day 220 Dec. 20, 2016 41

TABLE 26 RAPD fingerprint diversity of the Clostridium perfringens isolates over time from Wisconsin Farm E. Shannon-Wiener Isolates Clusters Index of Diversity Pretreatment 134 42 3.33 Treatment Day 60 282 39 2.83 Treatment Day 220 280 16 1.78

TABLE 27 Diversity of non-toxigenic Clostridium species and change in proportions of the two most predominant groups over time. Shannon- Wiener Clostridium Clostridium Species Iso- Spe- Index of beijerinckii bifermentans Diversity lates cies Diversity group group Pretreatment 73 4 0.22 96%   0% Treatment Day 60 20 8 1.40 5% 60% Treatment Day 13 3 0.93 0% 62% 220

Example 14: The Effect of a Combination of Bacillus Strains on the Clostridial Populations of Dairy Cows on Farm BS-TX in Texas

Introduction:

Hemorrhagic bowel syndrome (HBS) was first reported in 1991 and observed in five high-producing Holstein cows from one dairy in Idaho (Sockett, 2004). Symptoms included point-source sub-mucosal hematomas, each affecting 10-20 cm of the jejunum. One of the five cows exhibited a ruptured hematoma with exsanguination into the lumen of the jejunum. Although Aspergillus fumigatus and Clostridium perfringens are known to be involved in the etiology of HBS, the syndrome is better described as being poly-microbial and multi-factorial in nature. Increased consumption of a high-energy diet seems to be the most plausible common pathway for all the risk factors that have been described (Berghaus et al., 2005).

HBS is characterized by sudden drop in milk production, abdominal pain due to obstructed bowel and anemia (Anderson, 2002). Clinical signs of the disease are decreased feed intake, depression, decreased milk production, dehydration, abdominal distension and dark clotted blood in the feces. Death comes within 48 hours from the onset of the obstructing blood clot plug. Due to the sporadic, acute etiology few treatments are known to be effective

In addition to Clostridium isolates of several species causing enteric disease, other Clostridium species produce high levels of acetone, butanol, 1,3 propanediol and butyric acid as end products of their metabolism. If produced in the rumen, these metabolic end products may affect rumen function and decrease efficiency.

The Bacillus strains selected by the inventors, to inhibit pathogens, produce multiple compounds with inhibitory activity against other microbes with many strains containing more than ten operons producing antifungal and antibacterial compounds. Multiple bacteriocins are being produced in vitro directly at the site of action by the Bacillus strains so a robust blend of bacteriocins are present at doses lower than would be needed if isolated bacteriocins were being added directly to the feed.

The purpose of this study was to measure milk production and the levels and diversity of clostridia in dairy cows on Farm B-TX treated with the product, in accordance with this embodiment of the present invention, (referred to herein as “treated”), over 107 days.

Materials and Methods:

A dairy herd in Texas (Farm BS) was selected to study the impact of Bacillus product, in accordance with this embodiment of the present invention, on milk production and clostridia levels and diversity. The herd consists of 5500 milk cows housed in forced air-ventilated barns and the cows are bedded on sand.

The product, in accordance with this embodiment of the present invention was a combination product of two Bacillus strains in equal proportions; Bacillus 747 and Bacillus 1781 incorporated into the total mixed ration (TMR) at a dose of about 2 billion CFU per head per day.

The average Energy Corrected Milk (ECM) production was calculated for the 4 months prior to feeding the Bacillus to the dairy cows. The ECM production was calculated for the 4 months after the inclusion of the product into the feed. ECM is a calculation to standardize volume of milk produced on a total energy basis considering fluid milk production (lbs), milk fat (lbs), and milk protein (lbs). The calculation adjusts actual milk fat (lbs) to a standardized 3.5 percent and actual milk protein (lbs) to a standardized 3.2 percent. This calculation allows producers to compare the volume of milk produced on a standardized basis.

The formula for ECM:

ECM=(0.327×milk pounds)+(12.95×fat pounds)+(7.65×protein pounds)

Fecal samples were obtained from 60 cows before treatment and 60 cows after 107 days on treatment.

Fecal samples from cows were diluted 1:10 with sterile peptone, heat shocked for 30 minutes at 60° C., enumerated in sterile peptone and pour plated on Tryptose Sulphite Cycloserine (TSC) agar with D-cycloserine (400 mg/L) to select for clostridial species. Agar plates were incubated at 37° C. anaerobically for 24 hours. If present, isolated sulphite-reducing colonies were counted for total clostridia counts (CFU/g) and representative isolates were picked into Reinforced Clostridia Medium (RCM) (Oxoid, CM0149) and incubated anaerobically for 24 hours at 37° C. After 24 hours of incubation the cultures were transferred (10%) to Brain Heart Infusion (BHI) broth (BD, 211059) and incubated anaerobically for 24 hours at 37° C.

DNA extractions were performed in 96-well blocks containing 500 μl presumptive clostridia culture per well. Cells were harvested by centrifugation at 4,700 rpm for 10 minutes, the supernatant was removed. Cells were re-suspended in 500 μl of 50 mM of EDTA-2Na (pH=8.0). Aliquots of 300 μl of the suspended cells were transferred to a new 96-well block and combined with 20 μl of lysozyme from chicken egg white (Sigma, L6876) solution (100 mg/ml in 50 mM EDTA) to lyse bacterial cells. The 96-well block was incubated for 1 hour at 37° C. to lyse bacterial cells. Following the incubation 220 μl of lysis buffer (6 M Guanidine, 20% Triton-X 100, 10 mM Tris-HCL, pH 7.5) was added, mixed then incubated at room temperature for 15 minutes. Following the incubation 20 μl of Proteinase K (NEB, 800 U/mL) was added to each well, mixed and incubated at 55° C. for 30 minutes to degrade proteins. The cell lysate was then transferred to 96-well binding plate (Promega, A2278) and centrifuged at 4,700 rpms for 5 minutes. Flow through was discarded, three washes of the binding plate columns were executed centrifuging 750 μl of Column Wash Solution (Promega, A1318) at 4700 rpms for 1 minute and 30 seconds discarding flow through at the end of each spin. The binding plate was centrifuged for an additional 10 minutes at 4,700 rpm to remove any residual ethanol. A clean elution plate was then placed under the binding plate and DNA was eluted with 200 μl, pre-warmed (55° C.), Nuclease Free Water (Promega, P1195).

DNA was screened for toxin genes (α, β, ε, and ι) specific to C. perfringens using polymerase chain reaction (PCR). Amplification of toxin genes was executed using a multiplex PCR containing four primer sets (Yoo et al., 1997) (Table 1.) The PCR mixture contained 2.5 μl 10×PCR Buffer, 2 μl of 50 mM MgCl₂, 0.5 μM of each primer (Table 1.), 0.1 μl of Invitrogen™ Platinum™ Taq DNA Polymerase, 2.5 μl of DNA, sterile water was added to achieve 25 μl for a total reaction volume. The mixture underwent 5 minutes at 94° C., followed by 30 cycles of 94° C. for 1 minute, 55° C. for 1 minute, 72° C. for 1 minute finishing with a final elongation of 3 minutes at 72° C. PCR products were observed using a Fragment Analyzer (Advanced Analytics) to determine if amplification was achieved. If one or multiple toxin genes were observed a toxin type identification was assigned to each isolate based on their toxin-gene profile (Songer, 1996). C. perfringens positive to total clostridia isolate ratio was used to calculate an estimated C. perfringens count based on the total clostridia count.

Unique strain-specific genetic fingerprints were generated using Random Amplification of Polymorphic DNA (RAPD) analysis on select isolates to determine diversity among fecal C. perfringens isolates. The PCR contained 5 μl of DNA, 2.5 μl RAPD primer 2 (10 μM) (Table 1.), and 17.5 μl of sterile water which was added to a Ready-To-Go RAPD Analysis Bead (Life Sciences, 27-9500-01). The mixture underwent 5 minutes at 95° C., followed by 45 cycles of 95° C. for 1 minute, 36° C. for 1 minute, 72° C. for 2 minutes finishing with a final elongation of 5 minutes at 72° C. PCR products observed on a Fragment Analyzer (Advanced Analytics) to determine amplification patterns and were imported into BioNumerics, bioinformatics software, for analysis. RAPD patterns were compared with a band based Dice correlation analysis method to determine the similarity between RAPD patterns as a way to monitor diversity between isolates. Cluster cut-off was at 75% similarity.

To identify clostridia that did not have at least one toxin gene specific to C. perfringens, a PCR reaction was performed on the isolate DNA to amplify the 16S region of rDNA using primers 27F-YM and 1492R-Y (Table 1). This was done on 20% of the isolates that did not contain a toxin gene specific to C. perfringens. The PCR mixture contained 5 μl of 10×PCR Buffer, 2 μl of 50 mM MgCl₂, 1 μl of 50 mM dNTPs, 0.4 μM of each primer (Table 1.), 0.2 μl of Invitrogen™ Platinum™ Taq DNA Polymerase, 5 μl of DNA, and sterile water was added to achieve 50 μl for a total reaction volume. The mixture underwent 4 minutes at 95° C., followed by 35 cycles of 95° C. for 30 seconds, 50° C. for 30 seconds, 72° C. for 2 minutes finishing with a final elongation of 7 minutes at 72° C. A quality check was done on the amplification and PCR product was sent to Genewiz (https://www.genewiz.com) to obtain the sequences for the 16S genes. Sequences were compared to known type strains obtained from EZbiocloud online electronic database (http://www.ezbiocloud.net/). Based on comparisons of these sequences a bacterial identification was assigned to the isolates.

Results:

Milk production increased during the treatment period on Farm BS as ECM increased 0.6 lbs per day.

The average total clostridia counts on Farm BS (FIG. 89) were significantly higher (P>0.05) than those determined during the Texas survey (Example 6). There was no change in total clostridia or C. perfringens during treatment with the Bacillus blend (FIG. 90), however there was less strain diversity in the C. perfringens isolated during treatment as indicated by the Shannon-Wiever index of the C. perfringens RAPD patterns (Table 28). C. beijerincki group isolates were supplanted by C. bifermentans group isolates during treatment (FIG. 91 and Table 29).

Discussion:

Milk production improved during the treatment period, in addition the blend of Bacillus strains, in accordance with this embodiment of the present invention, decreased the diversity of C. perfringens strains in the cows. The Bacillus product also reduced the diversity of non-toxigenic clostridial species and caused the displacement of C. beijerinckii group strains by C. bifermentans. These data demonstrate that even when clostridial counts are not greatly reduced by the Bacillus strains the diversity of C. perfringens isolates and the types of Clostridium species are effected by the product. The reduction in the proportion of the C. beijerinckii group, which are known to produce high levels of butanol and acetone, most likely improved rumen fermentation and feed efficiency thereby increasing milk production as measured in these dairy cows.

TABLE 28 RAPD fingerprint diversity of the Clostridium perfringens isolates over time from Texas Farm BS. Shannon-Wiener Isolates Clusters Index of Diversity Pretreatment 190 39 2.94 Treatment Day 107 358 26 2.14

TABLE 29 Diversity of non-toxigenic Clostridium species and change in proportions of the two most predominant groups over time from Texas Farm BS. Shannon- Wiener Clostridium Clostridium Species Iso- Spe- Index of beijerinckii bifermentans Diversity lates cies Diversity group group Pretreatment 28 11 2.01 39%  4% Day 107 31 12 2.21  3% 29%

Example 15: Immune Gene Expression Response of Bacillus Strains to LPS Challenge in an Intestinal Epithelial Cell Line

Introduction:

An in vitro cell culture screening study was conducted to determine the effects of Bacillus strains and a combination of strains on the inflammatory cytokine gene expression response in an intestinal epithelial cell line. Bacillus strains were screened in the IEC-6 rat intestinal epithelial cell line in an LPS challenge model to determine the effect of each Bacillus strain and combination on inflammatory cytokine gene expression response with and without LPS challenge. Bacillus strains and combinations tested in the model included:

Bacillus strain 747

Bacillus strain 1781

Bacillus strain 1104

Bacillus strain 1541

Bacillus strain 2018

Bacillus strain 747+1781

Materials and Methods:

Cell Culture Preparation: The IEC6 rat intestinal epithelial cell line (ATCC) was used in this study; cells were expanded in a 37° C. incubator at 5% CO₂ to passage 18 in tissue culture flasks to 80% confluence in CCM Complete Growth Medium (DMEM: Dulbecco's Modified Eagle's Medium containing 4 mM L-glutamine and 4.5 g/L glucose; 1.5 g/L sodium bicarbonate; 0.1 Unit/mL bovine insulin, 90%; 10% FBS: fetal bovine serum) with 1% antibiotic (penicillin, 10 U/mL/streptomycin, 10 mg/mL). Growth media was removed from flasks and cells were then washed twice with warmed Dulbecco's Phosphate-Buffered Saline (DPBS). Trypsin warmed to 37° C. was added to cover the cell monolayer in the flask and incubated at 37° C. until cells lifted off the surface of the flask. CCM Growth Media was immediately added to the trypsinized cells, mixed thoroughly by pipetting and removed to a 15 mL conical tube. Cells were pelleted by centrifuging at 1500 rpm for 10 min. After discarding the supernatant, cells were resuspended in 2-3 mL CCM Growth Medium and cell concentration and viability was determined by diluting a portion of the cell suspension in Trypan Blue and counting on a hemocytometer. Cells were diluted to 3×10⁵ cells/mL, 1 mL of this cell suspension was added to each well of a 24-well plate, and incubated overnight in a 37° C. incubator at 5% CO₂ in order for cells to adhere to the bottom of the wells and form a monolayer. The following day, growth media was removed from each well, the cell monolayers were washed twice with DPBS, and treatments were administered to designated wells.

Cell Culture Treatment Administration:

Treatments were administered to wells in a 24-well. Antibiotic-free CCM media was used for this stage of the assay when treatments were administered. A 30 ng/mL LPS solution was prepared in CCM media without antibiotics and 500 uL of this solution was administered to each LPS treated well, resulting in 15 ng administered to each appropriate well. Bacillus strains were grown overnight in tryptic soy both and diluted in DPBS such that 250 uL of each respective treatment was administered to each well to deliver 1×10⁵ cfu/well. A total volume of 1 mL was administered to each well with either DPBS or CCM media added as appropriate to bring each well to an equal 1 mL volume. Specific volumes administered to Unstimulated, LPS, Bacillus, and Bacillus+LPS designated wells are shown in Table 30.

Following treatment administration to designated wells, plates were incubated for 1 hour at 37° C. and 5% CO₂. After the one hour incubation, media was removed from the wells and cells were washed 2× with warm DPBS. Immediately after the second DPBS wash, 400 uL of Trizol was added to each well and allowed to incubate at room temperature for 5 minutes. Trizol was removed from each well, placed in a 1.5 mL microcentrifuge tube labelled with designated treatment and immediately snap-frozen in liquid nitrogen. Tubes were stored in −80° C. for future RNA extraction and qPCR gene expression analysis.

RNA Extraction and cDNA Synthesis:

Frozen Trizol tubes were removed from freezer storage and allowed to thaw on ice. RNA was extracted using the Direct-zol RNA MiniPrep kit (Zymo Research, Irvine, Calif.) according to manufacturer's instructions. RNA concentration was checked on the Qubit fluorometer (ThermoFisher Scientific, Inc.) and recorded to document RNA extraction from each well of the cell culture experimental template. The QuantiNova Reverse Transcription kit (Qiagen, Germantown, Md.) was used to prepare cDNA per manufacturer's instructions.

Real-Time Ouantitative PCR:

qPCR was performed on the Applied Biosystems StepOne Plus Real Time PCR system (Applied Biosystems, Foster City, Calif.) using Platinum Taq polymerase and the primers listed in Table 31. β-Actin was used as the housekeeping gene and ΔCt values were determined for each well treatment by subtracting the number of PCR cycles related to the target gene from the PCR cycles associated with the housekeeping gene. Fold change in gene expression was also calculated using the ΔΔCt method to calculate fold change in gene expression of each LPS and Bacillus treatment relative to the unstimulated cells.

Results:

All Bacillus strains tested elicited some inflammatory response when exposed to the intestinal epithelial cell line, as indicated by the increased fold-change in gene expression of the inflammatory cytokines, MIP-2 and TNF-α (FIGS. 92-96). Of the five Bacillus strains tested, Bacillus 747 (FIG. 92) exhibited substantially lower fold increase in expression of the inflammatory cytokine genes than the others. When combined with LPS, three of the Bacillus strains (Bacillus 1781, Bacillus 1104, and Bacillus 2018; FIGS. 93, 94, and 96, respectively) reduced the inflammatory cytokine gene expression compared to LPS inflammatory challenge alone, indicating these Bacillus strains have the ability to ameliorate responses to inflammatory challenge in the gastrointestinal tissue. The combination of Bacillus 747 and Bacillus 1781 administered to the IEC-6 epithelial cell line resulted in a fold-change in gene expression similar to Bacillus 747 administered alone, and exhibited a moderate propensity to reduce the inflammatory response induced by LPS challenge, similarly to Bacillus 1781 (FIG. 97).

Discussion:

These data illustrate the immunomodulatory potential of Bacillus strains in an inflammatory challenge model, simulating the effects of a gram-negative bacterial pathogenic infection. Furthermore, individual Bacillus strains elicit distinct immunomodulatory effects on inflammation, suggesting Bacillus strains should for the specific functions (inflammatory or anti-inflammatory) to meet the health needs of the host.

TABLE 30 Primer sequences used in the cell culture yeast products study. Name Sequence β-Actin-F TGACGAGGCCCAGAGCAAGA (SEQ ID No. 18) β-Actin-R ATGGGCACAGTGTGGGTGAC (SEQ ID No. 19) MIP2-3F CCCCTTGGTTCAGAGGATCG (SEQ ID No. 20) MIP2-3R TTGATTCTGCCCGTTGAGGT (SEQ ID No. 21) TNFα-3F GGCCCGAGGCAACACAT (SEQ ID No. 22) TNFα-3R GGGCCATGGAACTGATGAGA (SEQ ID No. 23)

TABLE 31 Volumes administered to each well based on treatment designation. UNSTIM LPS Bacillus Bacillus + LPS CCM (w/o Ab) 750 uL 250 uL 750 uL 250 uL DPBS 250 uL 250 uL — — LPS — 500 uL — 500 uL Bacillus — — 250 uL 250 uL

Example 16: Administration of a Direct-Fed Microbial Containing Bacillus and Lactobacillus Strains to Calf Milk Replacer

Introduction:

Pre- and post weaning performance and health of nursery dairy calves when fed direct-fed microbials (DFM) supplemented into the milk replacers

Material and Methods:

A total of 100 Holstein heifer calves (39.2±0.65 kg body weight) were included in a trial to assess the effect of two different microbial combinations administered as a direct-fed microbial (DFM) added to calf milk replacer. The 56-day study included a 42-day pre-weaning portion in which calves were administered a 20/20 (20% fat/20% protein) milk replacer and a 14-day post-weaning period in which calves were fed completely on a dry diet. An all-milk protein, non-medicated milk replacer was fed at 0.28 kg in 2 L of water 2× daily from d 1 to d 35 and 1× daily from d 36 to weaning at d 42. The nutrient composition of milk replacer and dry feed used in the study are summarized in Table 32. Calves were randomly assigned to one of four treatments (25 calves/treatment) that were added to the daily mix of calf milk replacer administered individually to each calf on test:

1) Control—20/20 milk replacer

2) Antibiotic—Control supplemented with neomycin and oxytetracycline at a rate of 22 mg/kg BW for 14 days

3) Bacillus 747—Control supplemented with 5 g of DFM premix containing Bacillus strain 747 (1×10⁹ CFU/head/d) per feeding for 42 days

4) Bacillus 747+1781—Control supplemented with 5 g of DFM premix containing Bacillus strains 747+1781 (1×10⁹ CFU/head/d total CFU with each Bacillus strain representing 50% of the total) feeding for 42 days.

Body weight of calves was recorded on day 14, 28, 42, and 56 of the study and average daily gain (ADG) was calculated. Hip height was measured and recorded for each calf on day 1 and day 56 of the study. Intake of all feed was recorded daily and summarized every two weeks by treatment. Fecal scores were conducted on calves daily and summarized weekly through the first four weeks of the study, using a 1-4 scale defined as 1=normal, 2=loose, 3 very loose, but no watery separation, and 4=very watery. Health records including medication treatments, number of treatment days, medication treatment costs, and mortality were recorded throughout the study.

A 10 mL blood sample was obtained into EDTA tubes from each calf 14 days following the initial treatment administration. A 0.5 mL subsample was removed from the 10 mL sample and placed in RNA Protect tubes (Qiagen, Inc., Valencia, Calif.). Blood plasma was collected from the remaining whole blood sample and analyzed for the acute phase protein, haptoglobin. Haptoglobin was analyzed by ELISA kit per the manufacturer's instructions (MyBioSource, San Diego, Calif.). The blood from the RNA Protect tubes was used to extract RNA from blood cells and measure the gene expression analysis of various immune cytokines between the four treatments. Briefly, RNA extraction was performed on the blood sample stored in the RNA Protect tubes using the RNEasy Protect Animal Blood Kit per the manufacturer's instructions (Qiagen, Inc.). The QuantiNova Reverse Transcription kit (Qiagen, Germantown, Md.) was used to prepare cDNA per manufacturer's instructions. Real-time quantitative PCR (RT-qPCR) was performed on the Applied Biosystems StepOne Plus Real Time PCR system (Applied Biosystems, Foster City, Calif.) using Platinum Taq polymerase and the primers listed in Table 33. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene and ΔCt values were determined for each well treatment by subtracting the number of PCR cycles related to the target gene from the PCR cycles associated with the housekeeping gene.

Growth performance and calf health data were analyzed using the PROC MIXED procedure of SAS and repeated measures analyses applied where appropriate. Serum acute phase proteins and cytokine gene expression data were analyzed using the General Linear Model procedure using JMP. Least squares means were used to differentiate treatment effects using Student's t-test. Initial body weight (BW) was used as a covariate for BW, ADG and dry matter intake (DMI) data when significant. Initial hip heights were used as a covariate for day 56 hip height and hip height gain.

Results:

Calves fed the Antibiotic treatment (TRT2) and Bacillus 747 (TRT3) had greater (P=0.05) body weight on day 56 of the study and tended (P<0.10) to have greater ADG and total body weight gain compared to calves fed the Control (TRT1) milk replacer, whereas calves administered Bacillus 747+1781 (TRT4) was intermediate between the Control and the other treatments (Table 34). Milk replacer and calf starter feed intake were similar across all treatments (Table 35 and Table 36), and no differences were observed in serum proteins, fecal scores, scouring days, or treatment costs (Table 37).

Plasma haptoglobin concentrations were similar across all four treatments (FIG. 98). Gene expression of the reactive oxygen compound, iNOS, was greater (P<0.05) in the blood cells of calves administered the Bacillus 747+1781 treatment compared to all others (Table 38). Furthermore, gene expression of the inflammatory cytokine, IL-6, was greater (P<0.05) in calves fed the Antibiotic treatment compared to calves administered the Control and Bacillus 747 treatments, and calves administered the Control milk replacer and with Bacillus 747+1781 supplementation had greater (P<0.05) gene expression of the chemokine, CCL8, compared to calves fed the Antibiotic or Bacillus 747. The differences in cytokine gene expression observed in this study indicate that Bacillus 747 administered alone promotes a less inflammatory environment compared to the combination of Bacillus 747+178 1, as indicated by the greater gene expression of iNOS and CCL8 observed in calves fed Bacillus 747+1781.

Discussion:

Calves fed milk replacer with Bacillus supplementation had similar growth and health as calves offered milk replacers containing the antibiotics, neomycin sulfate:oxytetracycline for 14 days, indicating that Bacillus administered to calves has the potential to be used as alternatives to antibiotic administration to promote health and efficient growth. Furthermore, this study shows that specific Bacillus strains alone or in combination, elicit different immunomodulatory activities, promoting either an inflammatory or quiescent immunological environment in the young calf, potentially resulting in a divergence of immune development and function.

TABLE 32 Nutrient composition of basal milk replacer (MR) and basal calf starter (CS). Nutrient Analyses MR CS Dry Matter, % 96.14 86.31 ADF¹, % — 10.22 NDF¹, % 0.25 16.97 CP¹, % 21.34 19.72 Ash, % 10.15 6.61 Ether Extract, % 20.35 3.95 Starch, % — 35.98 Calcium, % 0.90 1.08 Phosphorus, % 0.91 0.63 Potassium, % 2.72 1.31 Magnesium, % 0.20 0.31 TDN¹, % — 76.77 NFC¹, % 47.92 53.53 ¹ADF = acid detergent fiber; NDF = neutral detergent fiber; CP = crude protein; TDN = total digestible nutrients; NFC = non-fiber carbohydrates

TABLE 33 Bovine primer sequences for immune gene expression analysis using RT-qPCR. Product Gene Forward Reverse Size PTX3 GGCAGACTCACAGGCTTCAATATC CCTTCTCCAGTCTCCCTTTCAACT 343 (SEQ. ID No. 24) GAPDH GGCGTGAACCACGAGAAGTATAA CCCTCCACGATGCCAAAGT 194 (SEQ. ID No. 25) SOD ACTTCGAGGCAAAGGGAGATAC TTTTGGCCCACCGTGTT 164 (SEQ. ID No. 26) COX2 TCCTGAAACCCACTCCCAACA TGGGCAGTCATCAGGCACAG 242 (SEQ. ID No. 27) iNOS GGCTACGGAACTGGACATCAAC CTCAGGGATTCTGGAGACG 162 (SEQ. ID No. 28) IL6 ATGACTTCTGCTTTCCCTACCC GCTGCTTTCACACTCATCATTC 179 (SEQ. ID No. 29) IL-18 CACGTTTCCTCTCCTAAGAAGC TACTTGTTCTGCAGCCATCTTT 60 (SEQ. ID No. 30) CCL2 CCAGATGCAATTAACTCCCAAG GCATGGAGATCTTCTTACTGTTGA 64 (SEQ. ID No. 31) CCL8 AAGCAGAAGTGGGTCCAGACT CTTCGGTGTTCGGGACTTT 60 (SEQ. ID No. 32) TLR-2 TCCACGGACTGTGGTACATGAAGA GCTTAAAGGGAGGGTTGAAGTGCT 180 (SEQ. ID No. 33) TLR-4 TCAGAGTTTCCTGCAGTGGGTCAA ACTAAGCACTGGCATGTCCTCCAT 106 (SEQ. ID No. 34)

TABLE 34 Growth parameters of calves measured on day 1 to 56 of the study. TRT1¹ TRT2¹ TRT3¹ TRT4¹ SEM Calves, n 25 25 25 25 Initial Body Weight², 85.6 85.1 85.4 85.5 1.37 lb Initial Hip Height³, in 31.48 31.79 31.66 31.47 0.18 Body Weight (BW), Lb d 14 93.0 95.1 93.8 93.2 0.72 d 28 105.4 108.0 107.6 105.9 1.10 d 42 123.5 126.8 127.4 124.3 1.75 d 56 149.1^(c) 155.7^(a) 154.8^(ab) 151.5^(a,b,c) 2.43 d 84 202.3 209.4 210.0 203.3 3.75 Average Daily Gain (ADG), lb/d d 1 to 14 0.54 0.69 0.59 0.55 0.04 d 15 to 28 0.90 0.94 1.00 0.84 0.06 d 29 to 42 1.30 1.36 1.43 1.40 0.09 d 1 to 42 0.90 0.98 0.99 0.91 0.05 d 43 to 56 1.85 2.07 1.97 1.96 0.09 d 1 to 56 1.15^(z) 1.26^(x) 1.25^(x,y) 1.19^(y,z) 0.04 d 57 to 84 1.89 1.90 1.95 1.84 0.07 d 1 to 84 1.39 1.47 1.48 1.4 0.04 Total BW Gain, lb d 1 to 14 7.5 9.6 8.3 7.7 0.63 d 15 to 28 12.6 13.1 14.0 11.8 0.89 d 29 to 42 18.3 19.0 20.0 19.5 1.20 d 1 to 42 37.8 41.1 41.7 38.3 1.97 d 43 to 56 25.8 29.0 27.6 27.4 1.21 d 1 to 56 64.2^(z) 70.7^(x) 69.8^(xy) 66.4^(y,z) 2.48 Hip Height, in d 56 35.18 35.4 35.3 35.43 0.14 d 84 37.57 37.84 37.69 38.04 0.18 Hip Height gain, in d 1 to d 56 3.59 3.80 3.70 3.83 0.14 d 57 to 84 2.41 2.44 2.39 2.61 0.14 d 1 to 84 5.98 6.25 6.09 6.44 0.18 ¹TRT1 = Control; TRT2 = Antibiotic; TRT3 = Bacillus 747; TRT4 = Bacillus 747 + 1781. ²Initial body weight (BW) utilized as a covariate for body weight and average daily gain data. ³Initial hip height (HH) utilized as a covariate for hip height measurement on day 56 and HH gain. ^(a,b,c)Means in the same row with different superscripts differ (P = 0.05). ^(x,y,z)Means in the same row with different superscripts differ (P < 0.10).

TABLE 35 Milk replacer (MR) intake of calves measured on day 1 to 42 of the study. TRT1¹ TRT2¹ TRT3¹ TRT4¹ SEM Total MR intake, lbs DM d 1 to 14 16.27 16.49 16.38 16.31 0.08 d 15 to 28 16.84 16.81 16.82 16.82 0.08 d 29 to 42 12.61 12.62 12.62 12.60 0.08 d 1 to 42 45.72 45.90 45.81 45.72 0.15 Intake, lb/d DM d 1 to 14 1.16 1.18 1.17 1.17 0.005 d 15 to 28 1.20 1.20 1.20 1.20 0.005 d 29 to 42 0.90 0.90 0.90 0.90 0.005 d 1 to 42 1.09 1.09 1.09 1.09 0.003 ¹TRT1 = Control; TRT2 = Antibiotic; TRT3 = Bacillus 747; TRT4 = Bacillus 747 + 1781.

TABLE 36 Starter feed, dry matter (DM) intake, and feed efficiency of calves measured on day 1 to 56 of the study. TRT1¹ TRT2¹ TRT3¹ TRT4¹ SEM Starter intake, lb/d d 1 to 7 0.02 0.02 0.02 0.01 0.01 d 8 to 14 0.03 0.05 0.04 0.04 0.01 d 15 to 21 0.22 0.26 0.26 0.19 0.05 d 22 to 28 0.43 0.52 0.62 0.44 0.07 d 29 to 35 0.87 0.93 0.98 0.82 0.09 d 36 to 42 1.83 1.96 2.12 1.86 0.12 d 43 to 49 3.44 3.54 3.70 3.56 0.17 d 50 to 56 4.12 4.48 4.46 4.30 0.14 Total starter intake, lbs d 1 to 42 23.75 26.35 28.32 23.47 2.45 d 43 to 56 53.71 57.09 57.91 55.53 2.44 d 1 to 56 76.62 82.34 85.36 78.57 4.00 DM Intake, lbs/d d 1 to 14 1.19 1.22 1.21 1.20 0.01 d 15 to 28 1.53 1.59 1.65 1.52 0.06 d 29 to 42 2.25 2.35 2.45 2.24 0.11 d 1 to 42 1.65 1.72 1.77 1.65 0.06 d 43 to 56 3.84 4.04 4.12 3.95 0.15 d 1 to 56 2.20 2.30 2.36 2.23 0.07 DM intake, lbs d 1 to 14 16.68 17.03 16.88 16.75 0.19 d 15 to 28 21.45 22.33 23.04 21.32 0.87 d 29 to 42 31.55 32.91 34.36 31.40 1.53 d 1 to 42 69.48 72.26 74.13 69.20 2.46 d 43 to 56 53.71 56.60 57.72 55.30 2.04 d 1 to 56 123.4 128.8 132.0 124.8 4.04 Gain:Feed Ratio d 1 to 14 0.44 0.55 0.48 0.45 0.03 d 15 to 28 0.57 0.58 0.59 0.53 0.03 d 29 to 42 0.57 0.57 0.57 0.62 0.02 d 1 to 42 0.54 0.57 0.56 0.55 0.01 d 43 to 56 0.49 0.52 0.47 0.50 0.02 d 1 to 56 0.52 0.55 0.53 0.52 0.01 ¹TRT1 = Control; TRT2 = Antibiotic; TRT3 = Bacillus 747; TRT4 = Bacillus 747 + 1781.

TABLE 37 Serum protein and health measurements of calves from d 1 to 56 of the study. TRT1¹ TRT2¹ TRT3¹ TRT4¹ SEM Serum Protein 5.96 5.87 5.87 5.87 0.15 Fecal Score² d 1 to 14 1.74 1.98 1.82 1.77 0.06 d 15 to 28 1.26 1.31 1.24 1.30 0.04 d 29 to 42 1.07 1.06 1.09 1.03 0.02 d 1 to 42 1.35 1.45 1.38 1.37 0.03 d 43 to 56 1.10 1.05 1.08 1.03 0.03 d 1 to 56 1.29 1.35 1.31 1.28 0.02 Scouring days³ d 1 to 42 2.71 3.26 2.38 3.08 0.36 d 43 to 56 0.00 0.06 0.00 0.03 0.03 # Days = 4 d 1 to 42 0.30 0.36 0.30 0.34 0.12 Treatment cost, $ d 1 to 42 0.41 0.43 0.70 0.62 0.19 d 43 to 56 0.00 0.00 0.15 0.00 0.18 d 1 to 56 0.34 0.37 0.85 0.56 0.29 ¹TRT1 = Control; TRT2 = Antibiotic; TRT3 = Bacillus 747; TRT4 = Bacillus 747 + 1781. ²Fecal score value from 1 to 4, with 1 = normal to 4 = watery. ³Scouring day = any day with a fecal score ≧3.

TABLE 38 Immune gene expression of calves 14 days after administration of milk replacer treatments. Immune Gene Expression, ΔCt² TRT1¹ TRT2¹ TRT3¹ TRT4¹ SEM P = PTX3 11.00 11.00 11.38 10.83 0.65 0.946 SOD 10.90 10.88 11.00 11.12 0.42 0.994 COX2 9.00 9.00 9.00 9.50 0.42 0.801 iNOS 13.20^(a) 11.56^(a,b) 13.12^(a) 11.00^(b) 0.71 0.094 IL6 20.00^(a) 14.29^(c) 19.20^(a,b) 16.25^(b,c) 1.21 0.004 IL-18 5.40 6.11 5.75 6.00 0.27 0.161 CCL2 11.00 11.13 11.75 11.00 0.98 0.354 CCL8 9.56^(b) 11.11^(a) 10.57^(a,b) 9.50^(b) 0.53 0.050 TLR-2 5.40 5.11 5.00 4.75 0.25 0.429 TLR-4 4.90 4.67 4.38 4.25 0.26 0.356 ¹TRT1 = Control; TRT2 = Antibiotic; TRT3 = Bacillus 747; TRT4 = Bacillus 747 + 1781. ²Note: ΔCt measures number of PCR cycles and is inversely proportional to gene expression; greater ΔCt = lower gene expression. ^(a,b,c)Means in the same row with different superscripts differ (P < 0.05).

Example 17: The Effect of a Combination of Bacillus Strains on the Herd Milk Production of Dairy Cows on Five Herds in Wisconsin

Introduction:

Hemorrhagic bowel syndrome (HBS) was first reported in 1991 and observed in five high-producing Holstein cows from one dairy in Idaho (Sockett, 2004). Symptoms included point-source sub-mucosal hematomas, each affecting 10-20 cm of the jejunum. One of the five cows exhibited a ruptured hematoma with exsanguination into the lumen of the jejunum. Although Aspergillus fumigatus and Clostridium perfringens are known to be involved in the etiology of HBS, the syndrome is better described as being poly-microbial and multi-factorial in nature. Increased consumption of a high-energy diet seems to be the most plausible common pathway for all the risk factors that have been described (Berghaus et al., 2005).

HBS is characterized by sudden drop in milk production, abdominal pain due to obstructed bowel and anemia (Anderson, 2002). Clinical signs of the disease are decreased feed intake, depression, decreased milk production, dehydration, abdominal distension, and dark clotted blood in the feces. Death comes within 48 hours from the onset of the obstructing blood clot plug. Due to the sporadic, acute etiology few treatments are known to be effective.

In addition to Clostridium isolates of several species causing enteric disease, other Clostridium species produce high levels of acetone, butanol, 1,3 propanediol and butyric acid as end products of their metabolism. If produced in the rumen, these metabolic end products may affect rumen function and decrease efficiency.

The Bacillus strains selected by the inventors, to inhibit pathogens, produce multiple compounds with inhibitory activity against other microbes with many strains containing more than ten operons producing antifungal and antibacterial compounds. Multiple bacteriocins are being produced in vitro directly at the site of action by the Bacillus strains so a robust blend of bacteriocins are present at doses lower than would be needed if isolated bacteriocins were being added directly to the feed.

The purpose of this study was to measure milk production in dairy cows on five farms in Wisconsin treated with the product, in accordance with this embodiment of the present invention.

Materials and Methods:

Five Wisconsin dairy farms were selected to study the impact of Bacillus, in accordance with this embodiment of the present invention, on Energy Corrected Milk (ECM). ECM is a calculation to standardize volume of milk produced on a total energy basis considering fluid milk production (lbs), milk fat (lbs), and milk protein (lbs). The calculation adjusts actual milk fat (lbs) to a standardized 3.5 percent and actual milk protein (lbs) to a standardized 3.2 percent. This calculation allows producers to compare the volume of milk produced on a standardized basis.

The formula for ECM:

ECM=(0.327×milk pounds)+(12.95×fat pounds)+(7.65×protein pounds)

Herd sizes in the study ranged from 185 head to 940 head. Farms selected for this summary are considered typical Wisconsin dairy farms. The five herds selected had no major management changes during the measured times.

The product, in accordance with this embodiment of the present invention was a combination product of three Bacillus strains; Bacillus 1104 (50%), Bacillus 1781 (45%) and Bacillus 1541 (5%) incorporated into the total mixed ration (TMR) at a dose of 2 billion CFU per head per day.

The average ECM production was calculated for the 4 months prior to feeding the Bacillus to the dairy cows. The Bacillus was included into the dairy cows feed ration on a daily basis (Bacillus Treatment Period). The ECM production was calculated for the 4 months after the inclusion of the Bacillus into the feed.

Results:

Herd responses (Table 39) ranged from an increase of 0.4 lbs ECM per day (Herd ID #2) to 2.8 lbs ECM per day (Herd ID #5). The average increase of ECM for all 5 herds is 1.82 lbs per day.

Discussion:

The blend of Bacillus strains, in accordance with this embodiment of the present invention, selected to inhibit clostridia, consistently improved milk production as measured by ECM across multiple farms of various sizes in Wisconsin.

TABLE 39 Improvement in Energy Corrected Milk (ECM) while on the Bacillus product from five herds in Wisconsin. Herd Herd Size Change in ID (cows) Pretreatment Bacillus Treatment ECM #1 806 Jan. 1, 2015 to Apr. 30, 2015 May 5, 2015 to Sep.1, 2015 +2.1 lbs #2 185 Feb. 15, 2015 to Jun. 4, 2015 Jun. 5, 2015 to Oct. 5, 2015 +0.4 lbs #3 940 May 5, 2015 to Sep. 14, 2015 Sep. 15, 2015 to Jan. 15, 2016 +2.6 lbs #4 317 Aug. 10 2015 to Dec. 9, 2015 Dec. 10, 2015 to Apr. 6, 2016 +1.2 lbs #5 264 Sep. 15, 2015 to Jan. 14, 2016 Jan. 15, 2016 to May 15, 2016 +2.8 lbs Average Increase of ECM +1.82 lbs 

Example 18: The Effect of a Combination of Bacillus Strains on the Herd Health, Milk Production and Clostridial Populations of Dairy Cows on Farm WB in Wisconsin

Introduction:

Hemorrhagic bowel syndrome (HBS) was first reported in 1991 and observed in five high-producing Holstein cows from one dairy in Idaho (Sockett, 2004). Symptoms included point-source sub-mucosal hematomas, each affecting 10-20 cm of the jejunum. One of the five cows exhibited a ruptured hematoma with exsanguination into the lumen of the jejunum. Although Aspergillus fumigatus and Clostridium perfringens are known to be involved in the etiology of HBS, the syndrome is better described as being poly-microbial and multi-factorial in nature. Increased consumption of a high-energy diet seems to be the most plausible common pathway for all the risk factors that have been described (Berghaus et al., 2005).

HBS is characterized by sudden drop in milk production, abdominal pain due to obstructed bowel and anemia (Anderson, 2002). Clinical signs of the disease are decreased feed intake, depression, decreased milk production, dehydration, abdominal distension and dark clotted blood in the feces. Death comes within 48 hours from the onset of the obstructing blood clot plug. Due to the sporadic, acute etiology few treatments are known to be effective.

In addition to Clostridium isolates of several species causing enteric disease, other Clostridium species produce high levels of acetone, butanol, 1,3 propanediol and butyric acid as end products of their metabolism. If produced in the rumen, these metabolic end products may affect rumen function and decrease efficiency.

The Bacillus strains selected by the inventors, to inhibit pathogens, produce multiple compounds with inhibitory activity against other microbes with many strains containing more than ten operons producing antifungal and antibacterial compounds. Multiple bacteriocins are being produced in vitro directly at the site of action by the Bacillus strains so a robust blend of bacteriocins are present at doses lower than would be needed if isolated bacteriocins were being added directly to the feed.

The purpose of this study was to measure the herd health, milk production, clostridia levels and diversity in dairy cows on Farm WB treated with the product, in accordance with this embodiment of the present invention, (referred to herein as “treated”), over 86 days. Treatment was discontinued for 98 days and then recommenced for a second period.

Materials and Methods:

A dairy herd in Wisconsin (Farm WB) was selected to study the impact of a Bacillus product, in accordance with this embodiment of the present invention, on herd health and milk production. The herd consists of 900 milk cows housed in a free stall barn and the cows are bedded on sand. The herd is milked 3 times daily and has a rolling herd average of approximately 30,000 pounds per cow per year.

The product, in accordance with this embodiment of the present invention was a combination product of three Bacillus strains in equal proportions; Bacillus 747, Bacillus 1781 and Bacillus 2018 incorporated into the total mixed ration (TMR) at a dose of 2 billion CFU per head per day.

Herd health was determined by measuring cow deaths due to digestive issues.

Milk production on the farm was tracked using Energy Corrected Milk (ECM). ECM is a calculation to standardize volume of milk produced on a total energy basis considering fluid milk production (lbs), milk fat (lbs), and milk protein (lbs). The calculation adjusts actual milk fat (lbs) to a standardized 3.5 percent and actual milk protein (lbs) to a standardized 3.2 percent. This calculation allows producers to compare the volume of milk produced on a standardized basis.

The formula for ECM:

ECM=(0.327×milk pounds)+(12.95×fat pounds)+(7.65×protein pounds)

Fecal samples were obtained from cows before, during and after treatment. Sample dates and number of animals sampled are indicated in Table 1.

Fecal samples from cows were diluted 1:10 with sterile peptone, heat shocked for 30 minutes at 60° C., enumerated in sterile peptone and pour plated on Tryptose Sulphite Cycloserine (TSC) agar with D-cycloserine (400 mg/L) to select for clostridial species. Agar plates were incubated at 37° C. anaerobically for 24 hours. If present, isolated sulphite-reducing colonies were counted for total clostridia counts (CFU/g) and representative isolates were picked into Reinforced Clostridia Medium (RCM) (Oxoid, CM0149) and incubated anaerobically for 24 hours at 37° C. After 24 hours of incubation the cultures were transferred (10%) to Brain Heart Infusion (BHI) broth (BD, 211059) and incubated anaerobically for 24 hours at 37° C.

DNA extractions were performed in 96-well blocks containing 500 μl presumptive clostridia culture per well. Cells were harvested by centrifugation at 4,700 rpm for 10 minutes, the supernatant was removed. Cells were re-suspended in 500 μl of 50 mM of EDTA-2Na (pH=8.0). Aliquots of 300 μl of the suspended cells were transferred to a new 96-well block and combined with 20 μl of lysozyme from chicken egg white (Sigma, L6876) solution (100 mg/ml in 50 mM EDTA) to lyse bacterial cells. The 96-well block was incubated for 1 hour at 37° C. to lyse bacterial cells. Following the incubation 220 μl of lysis buffer (6 M Guanidine, 20% Triton-X 100, 10 mM Tris-HCL, pH 7.5) was added, mixed then incubated at room temperature for 15 minutes. Following the incubation 20 μl of Proteinase K (NEB, 800 U/mL) was added to each well, mixed and incubated at 55° C. for 30 minutes to degrade proteins. The cell lysate was then transferred to 96-well binding plate (Promega, A2278) and centrifuged at 4,700 rpms for 5 minutes. Flow through was discarded, three washes of the binding plate columns were executed centrifuging 750 μl of Column Wash Solution (Promega, A1318) at 4700 rpms for 1 minute and 30 seconds discarding flow through at the end of each spin. The binding plate was centrifuged for an additional 10 minutes at 4,700 rpm to remove any residual ethanol. A clean elution plate was then placed under the binding plate and DNA was eluted with 200 μl, pre-warmed (55° C.), Nuclease Free Water (Promega, P1195).

DNA was screened for toxin genes (α, β, ε, and ι) specific to C. perfringens using polymerase chain reaction (PCR). Amplification of toxin genes was executed using a multiplex PCR containing four primer sets (Yoo et al., 1997) (Table 1.) The PCR mixture contained 2.5 μl 10×PCR Buffer, 2 μl of 50 mM MgCl₂, 0.5 μM of each primer (Table 1.), 0.1 μl of Invitrogen™ Platinum™ Taq DNA Polymerase, 2.5 μl of DNA, sterile water was added to achieve 25 μl for a total reaction volume. The mixture underwent 5 minutes at 94° C., followed by 30 cycles of 94° C. for 1 minute, 55° C. for 1 minute, 72° C. for 1 minute finishing with a final elongation of 3 minutes at 72° C. PCR products were observed using a Fragment Analyzer (Advanced Analytics) to determine if amplification was achieved. If one or multiple toxin genes were observed a toxin type identification was assigned to each isolate based on their toxin-gene profile (Songer, 1996). C. perfringens positive to total clostridia isolate ratio was used to calculate an estimated C. perfringens count based on the total clostridia count.

Unique strain-specific genetic fingerprints were generated using Random Amplification of Polymorphic DNA (RAPD) analysis on select isolates to determine diversity among fecal C. perfringens isolates. The PCR contained 5 μl of DNA, 2.5 μl RAPD primer 2 (10 μM) (Table 1.), and 17.5 μl of sterile water which was added to a Ready-To-Go RAPD Analysis Bead (Life Sciences, 27-9500-01). The mixture underwent 5 minutes at 95° C., followed by 45 cycles of 95° C. for 1 minute, 36° C. for 1 minute, 72° C. for 2 minutes finishing with a final elongation of 5 minutes at 72° C. PCR products observed on a Fragment Analyzer (Advanced Analytics) to determine amplification patterns and were imported into BioNumerics, bioinformatics software, for analysis. RAPD patterns were compared with a band based Dice correlation analysis method to determine the similarity between RAPD patterns as a way to monitor diversity between isolates. Cluster cut-off was at 75% similarity.

To identify clostridia that did not have at least one toxin gene specific to C. perfringens, a PCR reaction was performed on the isolate DNA to amplify the 16S region of rDNA using primers 27F-YM and 1492R-Y (Table 1). This was done on 20% of the isolates that did not contain a toxin gene specific to C. perfringens. The PCR mixture contained 5 μl of 10×PCR Buffer, 2 μl of 50 mM MgCl₂, 1 μl of 50 mM dNTPs, 0.4 μM of each primer (Table 1.), 0.2 μl of Invitrogen™ Platinum™ Taq DNA Polymerase, 5 μl of DNA, and sterile water was added to achieve 50 μl for a total reaction volume. The mixture underwent 4 minutes at 95° C., followed by 35 cycles of 95° C. for 30 seconds, 50° C. for 30 seconds, 72° C. for 2 minutes finishing with a final elongation of 7 minutes at 72° C. A quality check was done on the amplification and PCR product was sent to Genewiz (https://www.genewiz.com) to obtain the sequences for the 16S genes. Sequences were compared to known type strains obtained from EZbiocloud online electronic database (http://www.ezbiocloud.net/). Based on comparisons of these sequences a bacterial identification was assigned to the isolates.

Results:

The herd recorded 5 cow deaths due to digestive issues during the pretreatment period. The Bacillus product, in accordance with this embodiment of the present invention, was included into the dairy cows feed ration on a daily basis beginning 4-16-2016 and inclusion continued until 9-1-2016. During this Bacillus Treatment Period #1 the herd recorded one cow death due to digestive issues. The Bacillus was removed from the feed for a post-treatment period from 9-2-2016 to 12-8-2016. During this period the farm recorded 6 cow deaths due to digestive issues. The Bacillus Treatment Period #2 began on 12-9-2016 and the farm recorded zero cow deaths due to digestive issues thru 5-1-2017 (Table 41.).

Results indicate ECM increased 3.9 lbs/day and milk fat increased 0.3% during the Bacillus treatment period (Table 42.).

Average temperature was recorded from the time periods in 2015 and compared to the same time periods in 2016. This was done to ensure the positive milk response recorded during the Bacillus treatment Period was not simply due to a lower average temperature. The Bacillus treatment period in 2016 was an average of +2.6 F warmer on average in June and +2.7 F warmer in July (Table 43.).

Total clostridia counts on Farm WB (FIG. 99) were not significantly different than those determined during the Wisconsin survey (Example 5). During the first 16 days on the product total clostridia decreased significantly and then either were not different (days 30 and 86) or were higher (Day 58) compared to the Pretreatment. Clostridial counts for samples taken 98 days after the product was discontinued were significantly higher than before the product was started. Although there was no reduction in C. perfringens at any time during or after treatment with the Bacillus blend (FIG. 100), there was less strain diversity in the C. perfringens isolated during treatment and this was maintained post-treatment as indicated by the Shannon-Wiever index of the C. perfringens RAPD patterns (Table 44). Diversity of the non-toxigenic clostridia was also reduced during treatment and the predominant C. beijerinckii group isolates were supplanted by C. bifermentans group isolates during treatment (FIG. 101 and Table 45). When treatment was discontinued C. beijerinckii group strains became the predominant non-toxigenic clostridia again.

Discussion:

The blend of Bacillus strains, in accordance with this embodiment of the present invention, selected to inhibit clostridia, reduced the number of cow deaths due to digestive issues such as HBS. The number of digestive deaths increased again when the product was discontinued. Milk production as measured as ECM and milk fat was also improved. This production increase occurred even though more potential heat stress was present during the treatment period. The blend of Bacillus strains, in accordance with this embodiment of the present invention, selected to inhibit C. perfringens, initially caused a decrease in total clostridial counts and decreased the diversity of C. perfringens strains in the cows. There was also a reduction in the diversity of non-toxigenic clostridial species and the product caused the displacement of C. beijerinckii group strains by C. bifermentans. The proportions of C. beijerinckii group increased again when the treatment was discontinued. These data demonstrate that the product, in accordance with the embodiment of the present invention, improves herd health and milk production by reducing the levels and diversity of C. perfringens isolates and the diversity and types of Clostridium species present. The reduction in the proportion of the C. beijerinckii group, which are known to produce high levels of butanol and acetone, likely improves rumen fermentation and improves feed efficiency resulting in increased milk production in dairy cows.

TABLE 41 Number of cow deaths due to digestive issues from Wisconsin Farm WB. Dates Treatment Periods Deaths Jan. 15, 2016 to Apr. 15, 2016 Pretreatment 5 Apr. 16, 2016 to Sep. 1, 2016 Bacillus Treatment # 1 1 Sep. 2, 2016 to Dec. 8, 2016 Post-Treatment 6 Dec. 9, 2016 to May 1, 2017 Bacillus Treatment #2 0

TABLE 42 ECM production levels were tracked during a pretreatment period (Apr. 1, 2015 to Aug. 1, 2015) and a Bacillus treatment period (Apr. 1, 2016 to Aug. 1, 2016) from Wisconsin Farm WB. ECM Milk Fat Time Period Treatment Period (lbs) (%) Apr. 1, 2015 to Aug. 1, 2015 Pretreatment 98.3 3.9% Apr. 1, 2016 to Aug. 1, 2016 Bacillus Treatment # 1 102.2 4.2% Difference +3.9 +0.3%

TABLE 43 Weather Station Recordings- Temperature in Fahrenheit (F.) from Wisconsin Farm WB. June June June July July July Low High Average Low High Average 2015 55.2 76.2 65.7 58.6 79.7 69.1 2016 57.2 79.4 68.3 62.1 81.6 71.8 Difference +2.0 +3.2 +2.6 +3.5 +1.9 +2.7

TABLE 44 RAPD fingerprint diversity of the Clostridium perfringens isolates over time from Wisconsin Farm WB. Isolates Clusters Shannon-Wiener Pretreatment 316 81 3.93 Treatment Day 16 196 43 3.11 Treatment Day 30 145 33 3.02 Treatment Day 58 205 56 3.78 Treatment Day 86 328 64 3.60 Post-treatment 180 31 2.59

TABLE 45 Diversity of non-toxigenic Clostridium species and change in proportions of the two predominant groups over time for Wisconsin Farm WB. Shannon- Wiener Clostridium Clostridium Species Iso- Spe- Index of beijerinckii bifermentans Diversity lates cies Diversity group group Pretreatment 30 9 1.35 57% 13% Day 16 24 10 2.05  8% 42% Day 30 11 6 1.47 27%  9% Day 58 19 5 1.11 42% 32% Day 86 40 5 1.02 33% 58% Post-treatment 45 4 1.10 60% 16% Day 98

Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration from the specification and practice of the invention disclosed herein. All references cited herein for any reason, including all journal citations and U.S./foreign patents and patent applications, are specifically and entirely incorporated herein by reference. It is understood that the invention is not confined to the specific reagents, formulations, reaction conditions, etc., herein illustrated and described, but embraces such modified forms thereof as come within the scope of the following claims.

BIBLIOGRAPHY

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We claim:
 1. A direct fed microbial composition comprising an isolated Bacillus strain for use in reducing a Clostridium in a digestive system of a ruminant having ingested an effective amount of said direct fed microbial composition, wherein the isolated Bacillus strain is selected from the group consisting of: Bacillus 1104, Bacillus 1781, Bacillus 747, Bacillus 1541, Bacillus 1999, and Bacillus
 2018. 2. The direct fed microbial composition of claim 1, wherein the Clostridium is selected from a group consisting of: Clostridium perfringens, Clostridium bifermentans, Clostridium beijerinckii, Clostridium butyricum, Clostridium tertium, and Clostridium sordellii.
 3. The direct fed microbial composition of claim 2, wherein the Clostridium is Clostridium bifermentans, and wherein the isolated Bacillus strain inhibits the production of 1, 3-propanediol in the digestive system of the ruminant having ingested an effective amount of said direct fed microbial composition.
 4. The direct fed microbial composition of claim 2, wherein the Clostridium is Clostridium beijerinckii, and wherein the isolated Bacillus strain inhibits the production of butanol in the digestive system of the ruminant having ingested an effective amount of said direct fed microbial composition.
 5. The direct fed microbial composition of claim 2, wherein the Clostridium is Clostridium beijerinckii, and wherein the isolated Bacillus strain inhibits the production of acetone in the digestive system of the ruminant having ingested an effective amount of said direct fed microbial composition.
 6. The direct fed microbial composition of claim 2, wherein the Clostridium is Clostridium butyricum, and wherein the isolated Bacillus strain inhibits the excess production of butyrate in the digestive system of the ruminant having ingested an effective amount of said direct fed microbial composition.
 7. The direct fed microbial composition of claim 2, wherein the Clostridium is Clostridium perfringens, and wherein the isolated Bacillus strain reduces the occurrence of a digestive disorder in the ruminant having ingested an effective amount of said direct fed microbial composition.
 8. The direct fed microbial composition of claim 7, wherein the digestive disorder is hemorrhagic bowel syndrome.
 9. The direct fed microbial composition of claim 1, further comprising a cryoprotectant disposed about the isolated Bacillus strain, and wherein said isolated Bacillus strain is a biologically pure, powdered lyophilized strain.
 10. The direct fed microbial composition of claim 9, wherein the biologically pure, powdered lyophilized Bacillus strain comprises Bacillus spores.
 11. The direct fed microbial composition of claim 9, further comprising a carrier.
 12. The direct fed microbial composition of claim 1, wherein the effective amount of said direct fed microbial composition ingested by the ruminant per day comprises a concentration of the isolated Bacillus strain of between about 2×10⁸ CFU/ruminant and about 2.0×10¹⁰ CFU/ruminant.
 13. The direct fed microbial composition of claim 12, wherein the effective amount of said direct fed microbial composition ingested by the ruminant per day comprises a concentration of the isolated Bacillus strain of about 2×10⁹ CFU/ruminant.
 14. A method of improving ruminant performance, comprising: introducing into the digestive system of one or more ruminants an effective amount of the direct fed microbial composition according to claim 1; the introduction of the direct fed microbial providing at least one benefit chosen from: inhibiting a pathogen chosen from at least one of Clostridium perfringens, Clostridium bifermentans, Clostridium beijerinckii, Clostridium butyricum, Clostridium tertium, and Clostridium sordellii in the one or more ruminants; decreasing a mortality rate of the one or more ruminants; improving the feed efficiency of the one or more ruminants; reducing the occurrence of Hemorrhagic bowel syndrome in the one or more ruminants; improve rumen fermentation in the one or more ruminants; improve milk production in the one or more ruminants; and modulating immune responses of inflammatory cytokines in systemic and intestinal immune cells in the one or more ruminants.
 15. The method of claim 14, wherein the isolated Bacillus strains comprise Bacillus 1781 and Bacillus 747 and the direct fed microbial administration provides the benefit of decreasing diversity of Clostridium perfringens strains in the one or more ruminants.
 16. The method of claim 14, wherein the isolated Bacillus strains comprise Bacillus 1781 and Bacillus 747 and the direct fed microbial administration provides the benefit of decreasing diversity of non-toxigenic clostridial strains in the one or more ruminants.
 17. The method of claim 14, wherein the isolated Bacillus strains comprise Bacillus 1781 and Bacillus 747 and the direct fed microbial administration provides the benefit of increasing average energy corrected milk production in the one or more ruminants when the one or more ruminants are dairy cows.
 18. The method of claim 14, wherein the isolated Bacillus strains comprise Bacillus 1781, Bacillus 747, and Bacillus 2018 and the direct fed microbial administration provides the benefit of decreasing a digestive system related mortality rate of the one or more ruminants during a period of direct fed microbial administration.
 19. The method of claim 14, wherein the introduction step includes adding the direct fed microbial composition to a ruminant feed.
 20. A direct fed microbial composition comprising: a cryoprotectant disposed about a powdered lyophilized isolated Bacillus strain in the form of spores chosen from at least one of Bacillus 1104, Bacillus 1781, Bacillus 747, Bacillus 1541, Bacillus 1999, and Bacillus 2018; and a carrier, wherein the composition inhibits at least one pathogen selected from: Clostridium perfringens, Clostridium bifermentans, Clostridium beijerinckii, Clostridium butyricum, Clostridium tertium, and Clostridium sordellii in a digestion system of a ruminant having ingested an effective amount of said direct fed microbial composition, and wherein the effective amount of said direct fed microbial composition comprises a concentration of the isolated Bacillus strain of between about 2×10⁸ CFU/ruminant/day and about 2.0×10¹⁰ CFU/ruminant/day. 